Reagent storage in an assay device

ABSTRACT

The invention relates to methods for conducting binding assays in an assay device that includes one or more storage and use zone. The storage zones of the assay device are configured to house one or more reagents used in an assay conducted in the use zone of the device.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 15/206,656, filed Jul. 11, 2016, which is a continuation of U.S.patent application Ser. No. 13/272,350, filed Oct. 13, 2011, nowabandoned, which claims the benefit of U.S. Provisional Application No.61/455,112, filed Oct. 14, 2010, the disclosure of which is incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

Improved methods for conducting binding assays are provided. Thesemethods include a pre-concentration step to improve assay performance,for example, by increasing the concentration of analyte in the sampleand/or by reducing the concentration of extraneous materials that may bepresent in the sample which may hinder the performance of the bindingassay.

BACKGROUND OF THE INVENTION

A substantial body of literature has been developed concerningtechniques that employ binding reactions, e.g., antigen-antibodyreactions, nucleic acid hybridization and receptor-ligand reactions, forthe sensitive measurement of analytes of interest in samples. The highdegree of specificity in many biochemical binding systems has led tomany assay methods and systems of value in a variety of marketsincluding basic research, human and veterinary diagnostics,environmental monitoring and industrial testing. The presence of ananalyte of interest may be measured by directly measuring theparticipation of the analyte in a binding reaction. In some approaches,this participation may be indicated through the measurement of anobservable label attached to one or more of the binding materials.

There is always a desire to improve binding assays and devices used toconduct binding assays by increasing the signal obtained from a bindingevent, reducing non-specific binding, and/or improving measurementaccuracy and precision, especially when analyzing complex biologicalsamples.

SUMMARY OF TILE INVENTION

The invention provides an assay device including (a) a storage zonecomprising a surface-reagent complex confined to the storage zone, thesurface-reagent complex comprising (i) a reagent linked to a firsttargeting agent; and (ii) a surface linked to a second targeting agent,wherein the reagent and the surface are linked, in the surface-reagentcomplex, via a releasable binding interaction between the first andsecond targeting agents; and (b) one or more use zones each configuredto use the reagent in an assay for an analyte of interest in a sample.The assay device of the invention may include one or more storage zonesand/or one or more use zones. Additionally, the storage zone may alsoinclude two or more surface-reagent complexes, each including a distinctassay reagent that may be used in an assay conducted in the one or moreuse zones. For example, the storage zone also includes a secondsurface-reagent complex confined to the storage zone, the secondsurface-reagent complex comprising (i) a second reagent linked to athird targeting agent; and (ii) a second surface linked to a fourthtargeting agent, wherein the second reagent and the second surface arelinked, in the second surface-reagent complex, via a second releasablebinding interaction between the third and fourth targeting agents; andthe one or more use zones are further configured to use the secondreagent in the assay. The use zones may each comprise two or more assayregions each configured to use the reagent(s) stored in the storage zonein one or more assays conducted with a sample in the assay device.

The device may be used to conduct a plurality of assays for one or moreanalytes present in the sample, e.g., a first assay region of the one ormore use zones are each configured to conduct an assay for a firstanalyte of interest in the sample and an additional assay region in theone or more use zones is configured to conduct an assay for anadditional analyte of interest in the sample. Alternatively, a firstassay region of the one or more use zones is configured to conduct afirst assay for the analyte of interest in the sample and an additionalassay region of the one or more use zones is configured to conduct asecond assay for the analyte of interest in the sample.

The invention also provides a multiplexed assay device comprising (a) astorage zone comprising a surface-reagent complex confined to thestorage zone, the surface-reagent complex comprising (i) a reagentlinked to a first targeting agent; and (ii) a surface linked to a secondtargeting agent, wherein the reagent and the surface are linked, in thesurface-reagent complex, via a releasable binding interaction betweenthe first and second targeting agents; and (b) one or more use zoneseach comprising a plurality of assay regions configured to use thereagent in a multiplexed assay for a plurality of analytes in a sample.A first assay region of the one or more use zones is configured toconduct an assay for a first analyte of interest in the sample and anadditional assay region in the one or more use zones is configured toconduct an assay for an additional analyte of interest in the sample. Inaddition, the storage zone may further comprises a secondsurface-reagent complex confined to the storage zone, the secondsurface-reagent complex comprising (i) a second reagent linked to athird targeting agent; and (ii) a second surface linked to a fourthtargeting agent, wherein the second reagent and the second surface arelinked, in the second surface-reagent complex, via a second releasablebinding interaction between the third and fourth targeting agents; andthe one or more use zones are further configured to use the secondreagent in the multiplexed assay.

The invention further provides a method of conducting an assay in anassay device as described herein, including the steps: (x) introducingthe sample to the one or more use zones; (y) subjecting the storage zoneto a condition that releases the reagent from the surface-reagentcomplex; (z) transferring the reagent from the storage zone to the oneor more use zones; and (xx) conducting the assay in the one or more usezones with the reagent. If the use zones are each configured to use asecond reagent in an assay, the method further comprises, prior to theconducting step, subjecting the storage zone to an additional conditionthat releases the second reagent from the second surface-reagentcomplex; and transferring the second reagent from the storage zone tothe one or more use zones.

A method of using such an assay device may also include the steps of (x)introducing the sample to the one or more use zones; (i) subjecting thestorage zone to a condition that releases the reagent from thesurface-reagent complex; (ii) subjecting the storage zone to a conditionthat releases the second reagent from the second surface-reagentcomplex; (y) transferring the reagent from the storage zone to the firstassay region; (z) transferring the second reagent from the storage zoneto the second assay region; (xx) conducting an assay in the first assayregion with the reagent; and (yy) conducting an assay in the secondassay region with the second reagent. The transferring steps may besimultaneous or sequential. Similarly, the conducting steps may also besimultaneous or sequential.

In addition, the invention provides a method of using an assay device ofthe invention including the steps: (x) introducing the sample to the oneor more use zones; (y) subjecting the storage zone to a condition thatreleases the reagent from the surface-reagent complex; (z) transferringthe reagent from the storage zone to the first assay region and thesecond assay region; (xx) conducting the assays in the first and secondassay regions, respectively. The assays may be conducted simultaneouslyor sequentially.

In another embodiment, the assay device of the invention may be used inthe conduct of an assay by (x) introducing the sample to the one or moreuse zones via the storage zone; (y) adding a diluent to the storage zoneand (i) subjecting the storage zone to a condition that releases thereagent from the surface-reagent complex; (ii) subjecting the storagezone to an additional condition that releases the second reagent fromthe second surface-reagent complex; (z) transferring the reagent and thesecond reagent from the storage zone to the first and second assayregions; (xx) conducting the assays in the first and second assayregions. The assays and/or transfer steps may be conductedsimultaneously and/or sequentially.

Still further, the assay device may be used in an assay by (x)introducing the sample to the one or more use zones via the storagezone; (y) adding a diluent to the storage zone and (i) subjecting thestorage zone to a condition that releases the reagent from thesurface-reagent complex; (ii) subjecting the storage zone to anadditional condition that releases the second reagent from the secondsurface-reagent complex; (z) transferring the reagent from the storagezone to the first assay region; (xx) transferring the second reagentfrom the storage zone to the second assay region; (yy) conducting theassays in the first and second assay regions. The assays and/or transfersteps may be conducted simultaneously and/or sequentially.

Moreover, the invention provides a multiplexed assay device comprising(a) a storage zone comprising (i) a first reagent linked to a surface inthe storage zone via a first releasable binding interaction; (ii) asecond reagent linked to a second surface in the storage zone via asecond releasable binding interaction; (b) a first use zone configuredto use the first reagent in an assay for a first analyte; and (c) asecond use zone configured to use the second reagent in an assay for asecond analyte. The first releasable binding interaction comprises alinkage between a first targeting agent and a second targeting agent,wherein the first targeting agent is linked to the reagent and thesecond targeting agent is linked to the surface. Moreover, the reagentand the surface are linked to form a surface-reagent complex, whereinthe surface-reagent complex is confined to the storage zone. The secondreleasable binding interaction comprises a linkage between a thirdtargeting agent and a fourth targeting agent, wherein the thirdtargeting agent is linked to the second reagent and the fourth targetingagent is linked to the second surface, and the second reagent and thesecond surface are linked to form a second surface-reagent complex,wherein the second surface-reagent complex is confined to the storagezone. Such a multiplexed assay device comprises a fluidic network, suchthat the storage zone and the first and second use zones are in fluidiccommunication, wherein the network is configured to direct fluid in thestorage zone to the first use zone, the second use zone, or both. Thenetwork is configured to direct fluid to the first use zone and thesecond use zone sequentially or simultaneously. The first and secondreagents are each confined in the storage zone to distinct regions ofthe storage zone. The first and second releasable binding interactionsrequire the same or different conditions to release the first and secondreagents respectively, from the first and second surfaces of the storagezone, e.g., subjecting the storage zone to increased or decreasedtemperature, pH changes, an electric potential, a change in ionicstrength, competition, and combinations thereof. Moreover, each of thefirst and second use zones comprise a plurality of assay regions eachconfigured to use the first and second reagents in a multiplexed assayfor a plurality of different analytes in a sample.

Also provided is a method of conducting a multiplexed assay using themultiplexed assay device described herein including (a) introducing asample comprising the first and second analytes to the first and seconduse zones; (b) subjecting the storage zone to a condition that releasesthe first reagent from the storage zone; (c) transferring the firstreagent from the storage zone to at least one of the first and seconduse zones; and (d) conducting one or more assays for at least one of thefirst and second analytes.

The method may also include the steps of subjecting the storage zone toan additional condition that releases the second reagent from thestorage zone and transferring the second reagent from the storage zoneto at least one of the first and second use zones, and optionallywashing at least one of the first and second use zone prior to thetransferring step.

Also provided is a method of conducting a multiplexed assay in amultiplexed assay device including (a) introducing a sample comprisingthe first and second analytes to the first and second use zones; (b)subjecting the storage zone to a condition that releases the firstreagent from the storage zone; (c) transferring the first reagent fromthe storage zone to the first use zone; (d) subjecting the storage zoneto a condition that releases the second reagent from the storage zone;(e) transferring the second reagent from the storage zone to the seconduse zone; and (f) conducting assays for the first and second analytes inthe first and second use zones. The method may also include washing thefirst and second use zones prior to the transferring step (c), and theassays may be conducted simultaneously or sequentially.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are provided to illustrate rather than limitthe scope of the invention. Throughout the accompanying Figures, “P”refers to a particle to which one or more moieties are attached; “S”refers to a first solid phase; “A” refers to a target analyte; “C”refers to contaminants; and “*” refers to a detectable label linked toan assay component.

FIGS. 1(a)-1(e) illustrate various assay formats in which a particle isused as an assay component.

FIGS. 2(a)-2(b) illustrate various assay formats in which a first solidphase is used as an assay component.

FIGS. 3(a)-3(e) illustrate various assay formats in which a particle isused as an assay component, to which a targeting agent is linked.

FIGS. 4(a)-4(b) illustrate various assay formats in which a first solidphase is used as an assay component, to which a targeting agent islinked.

FIGS. 5(a)-5(b) illustrates one embodiment of the present invention.FIG. 5(a) shows magnetic concentration of analytes using colloids coatedwith anti-antibodies against the analytes and also coated with ECLlabels. Multiple antibodies may be used to bind different analytes. FIG.5(b) shows detection of the analyte-colloid complexes in a sandwichimmunoassay format.

FIGS. 6(a)-6(b) illustrate two alternative competitive immunoassaysaccording to the methods of the present invention.

FIG. 7(a)-7(c) illustrate three alternative embodiments of an assaydevice include one or more storage zones and one or more use zones.FIGS. 7(a)-(b) show an assay device including one storage zone thathouses a surface-reagent complex that supplies reagent to use zones 1and 2, while FIG. 7(c) shows an assay device including multiple storagezones that each lead to a use zone. In 7(c), sample and liquid reagentcompartments in the assay device are in fluid communication with thestorage and use zones.

FIGS. 8(a)-8(f) illustrate the use of an alternate assay device of thepresent invention.

FIGS. 9a-9e show non-scale schematic views of several embodiments ofmulti-well plate wells that include dry reagents.

FIGS. 10a-10j show non-scale schematic top and cross-sectional views ofseveral embodiments of wells having walls with shelf elements includingledges (FIGS. 10a-10f ), bridges (FIGS. 10g-10h ) and tables (FIGS.10i-10j ) that may be used to support dry reagents.

FIGS. 11a-11c show schematic illustrations of multi-well plates havingdetection wells and reagent reconstitution wells.

FIGS. 12a-12b show top and cross-sectional schematic views of oneembodiment of a plate having detection wells and reagent reconstitutionwells, the reagent reconstitution wells being located in interstitialspaces between the detection wells.

FIGS. 13a-13f show schematic views of multi-well plates 500 (FIGS.13a-13b ), 520 (FIGS. 13c-13d ) and 540 (FIGS. 13e-13f ) having assaywells and desiccant wells.

FIG. 14 is a schematic exploded view of one embodiment of a multi-wellassay plate.

FIGS. 15a-15c show three schematic views of a multi-well plate that isconfigured to carry out array-based multiplexed electrochemiluminescenceassays.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides improved solid phase binding assays thatinclude a collection, separation and/or release step. The methods of thepresent invention improve assay performance by allowing forpre-concentration of an analyte in a sample and/or by reducing oreliminating the amount of contaminants in a sample that may hinder theperformance of the assay, e.g., by interfering with the detection stepand/or by non-specifically binding with one or more of the components inthe mixture.

(i) Samples/Analytes

Examples of samples that may be analyzed by the methods of the presentinvention include, but are not limited to food samples (including foodextracts, food homogenates, beverages, etc.), environmental samples(e.g., soil samples, environmental sludges, collected environmentalaerosols, environmental wipes, water filtrates, etc.), industrialsamples (e.g., starting materials, products or intermediates from anindustrial production process), human clinical samples, veterinarysamples and other samples of biological origin. Biological samples thatmay be analyzed include, but are not limited to, feces, mucosal swabs,physiological fluids and/or samples containing suspensions of cells.Specific examples of biological samples include blood, serum, plasma,feces, mucosal swabs, tissue aspirates, tissue homogenates, cellcultures and cell culture supernatants (including cultures of eukaryoticand prokaryotic cells), urine, saliva, sputum, and cerebrospinal fluid.

Analytes that may be measured using the methods of the inventioninclude, but are not limited to proteins, toxins, nucleic acids,microorganisms, viruses, cells, fungi, spores, carbohydrates, lipids,glycoproteins, lipoproteins, polysaccharides, drugs, hormones, steroids,nutrients, metabolites and any modified derivative of the abovemolecules, or any complex comprising one or more of the above moleculesor combinations thereof. The level of an analyte of interest in a samplemay be indicative of a disease or disease condition or it may simplyindicate whether the patient was exposed to that analyte.

The assays of the present invention may be used to determine theconcentration of one or more, e.g., two or more analytes in a sample.Thus, two or more analytes may be measured in the same sample. Panels ofanalytes that can be measured in the same sample include, for example,panels of assays for analytes or activities associated with a diseasestate or physiological conditions. Certain such panels include panels ofcytokines and/or their receptors (e.g., one or more of TNF-alpha,TNF-beta, IL1-beta, IL2, IL4, IL6, IL-10, IL-12, IFN-y, etc.), growthfactors and/or their receptors (e.g., one or more of EGF, VGF, TGF,VEGF, etc.), drugs of abuse, therapeutic drugs, vitamins, pathogenspecific antibodies, auto-antibodies (e.g., one or more antibodiesdirected against the Sm, RNP, SS-A, SS-alpha, J0-1, and Scl-70antigens), allergen-specific antibodies, tumor markers (e.g., one ormore of CEA, PSA, CA-125 II, CA 15-3, CA 19-9, CA 72-4, CYFRA 21-1, NSE,AFP, etc.), markers of cardiac disease including congestive heartdisease and/or acute myocardial infarction (e.g., one or more ofTroponin T, Troponin I, myoglobin, CKMB, myeloperoxidase, glutathioneperoxidase, β-natriuretic protein (BNP), alpha-natriuretic protein(ANP), endothelin, aldosterone, C-reactive protein (CRP), etc.), markersassociated with hemostasis (e.g., one or more of Fibrin monomer,D-dimer, thrombin-antithrombin complex, prothrombin fragments 1 & 2,anti-Factor Xa, etc.), markers of acute viral hepatitis infection (e.g.,one or more of IgM antibody to hepatitis A virus, IgM antibody tohepatitis B core antigen, hepatitis B surface antigen, antibody tohepatitis C virus, etc.), markers of Alzheimers Disease (alpha-amyloid,beta-amyloid, Aβ 42, Aβ 40, Aβ 38, Aβ 39, Aβ 37, Aβ 34, tau-protein,etc.), markers of osteoporosis (e.g., one or more of cross-linked NorC-telopeptides, total deoxypyridinoline, free deoxypyridinoline,osteocalcin, alkaline phosphatase, C-terminal propeptide of type Icollagen, bone-specific alkaline phosphatase, etc.), markers offertility state or fertility associated disorders (e.g., one or more ofEstradiol, progesterone, follicle stimulating hormone (FSH), lutenizinghormone (LH), prolactin, hCG, testosterone, etc.), markers of thyroiddisorders (e.g., one or more of thyroid stimulating hormone (TSH), TotalT3, Free T3, Total T4, Free T4, and reverse T3), and markers ofprostrate cancer (e.g., one or more of total PSA, free PSA, complexedPSA, prostatic acid phosphatase, creatine kinase, etc.). Certainembodiments of invention include measuring, e.g., one or more, two ormore, four or more or 10 or more analytes associated with a specificdisease state or physiological condition (e.g., analytes groupedtogether in a panel, such as those listed above; e.g., a panel usefulfor the diagnosis of thyroid disorders may include e.g., one or more ofthyroid stimulating hormone (TSH), Total T3, Free T3, Total T4, Free T4,and reverse T3).

The methods of the present invention are designed to allow detection ofa wide variety of biological and biochemical agents, as described above.In one embodiment, the methods may be used to detect pathogenic and/orpotentially pathogenic virus, bacteria and toxins including biologicalwarfare agents (“BWAs”) in a variety of relevant clinical andenvironmental matrices, including and without limitation, blood, sputum,stool, filters, swabs, etc. A non-limiting list of pathogens and toxinsthat may be analyzed (alone or in combination) using the methods of thepresent invention is Bacillus anthracis (anthrax), Yersinia pestis(plague), Vibrio cholerae (cholera), Francisella tularensis (tularemia),Brucella spp. (Brucellosis), Coxiella burnetii (Q fever), orthopoxviruses including variola virus (smallpox), viral encephalitis,Venezuelan equine encephalitis virus (VEE), western equine encephalitisvirus (WEE), eastern equine encephalitis virus (EEE), Alphavirus, viralhemorrhagic fevers, Arenaviridae, Bunyaviridae, Filoviridae,Flaviviridae, Ebola virus, staphylococcal enterotoxins, ricin, botulinumtoxins, Clostridium botulinum, mycotoxin, Fusarium, Myrotecium,Cephalosporium, Trichoderma, Verticimonosporium, Stachybotrys, glanders,wheat fungus, Bacillus globigii, Serratia marcescens, yellow rain,trichothecene mycotoxins, Salmonella typhimurium, aflatoxin, Xenopsyllacheopis, Diamanus montanus, alastrim, monkeypox, Arenavirus, Hantavirus,Lassa fever, Argentine hemorrhagic fevers, Bolivian hemorrhagic fevers,Rift Valley fever virus, Crimean-Congo virus, Hanta virus, Marburghemorrhagic fevers, yellow fever virus, dengue fever viruses, influenza(including human and animal strains including H5N1 avian influenza),human immunodeficiency viruses I and II (HIV I and II), hepatitis A,hepatitis B, hepatitis C, hepatitis (non-A, B or C), Enterovirus,Epstein-Barr virus, Cytomegalovirus, herpes simplex viruses, Chlamydiatrachomatis, Neisseria gonorrheae, Trichomonas vaginalis, humanpapilloma virus, Treponema pallidum, Streptococcus pneumonia,Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae,Legionella pneumophila, Staphylococcus aureus, Moraxella catarrhalis,Streptococcus pyogenes, Clostridium difficile, Neisseria meningitidis,Klebsiella pneumoniae, Mycobacterium tuberculosis, coronavirus,Coxsackie A virus, rhinovirus, parainfluenza virus, respiratorysyncytial virus (RSV), metapneumovirus, and adenovirus.

(ii) Binding Reagents

The skilled artisan in the field of binding assays will readilyappreciate the scope of binding agents and companion binding partnersthat may be used in the present methods. A non-limiting list of suchpairs include (in either order) oligonucleotides and complements,receptor/ligand pairs, antibodies/antigens, natural or syntheticreceptor/ligand pairs, amines and carbonyl compounds (i.e., bindingthrough the formation of a Schiff's base), hapten/antibody pairs,antigen/antibody pairs, epitope/antibody pairs, mimitope/antibody pairs,aptamer/target molecule pairs, hybridization partners, andintercalater/target molecule pairs.

The binding assays of the methods of the present invention may employantibodies or other receptor proteins as binding reagents. The term“antibody” includes intact antibody molecules (including hybridantibodies assembled by in vitro re-association of antibody subunits),antibody fragments and recombinant protein constructs comprising anantigen binding domain of an antibody (as described, e.g., in Porter, R.R. and Weir, R. C. J. Cell Physiol., 67 (Suppl); 51-64 (1966) andHochman, 1. Inbar, D. and Givol, D. Biochemistry 12: 1130 (1973)), aswell as antibody constructs that have been chemically modified, e.g., bythe introduction of a detectable label.

Binding reagents and binding partners that are linked to assaycomponents to enable the attachment of these assay components to eachother or to solid phases may be described herein as “targeting agents”.For targeting agents that work in pairs, e.g., antigen-antibody,oligonucleotides-complement, etc., one targeting agent of the pair maybe referred to herein as the first targeting agent, whereas thecompanion targeting agent may be referred to as the second targetingagent. In certain embodiments, these targeting agents are selected basedon the reversibility of their binding reactions. In particular,targeting agent pairs may be selected, e.g., because under a first setof conditions the pair will bind to form a binding complex which, undera second set of conditions, can be caused to dissociate to break apartthe complex, e.g., by subjecting bound targeting agent pairs toincreased or decreased temperature, changes in chemical environment orassay buffer (e.g., ionic strength changes, pH changes, addition ofdenaturants, changes in light or electrical potential, etc.), addingcompeting binding reagents that compete with one targeting agent forbinding to another targeting agent, and combinations thereof. Suitableconditions may be derived through routine experimentation. There aremany well-established cleavable chemical linkers that may be used thatprovide a covalent bond that may be cleaved without requiring harshconditions. For example, disulfide containing linkers may be cleavedusing thiols or other reducing agents, cis-diol containing linkers maybe cleaved using periodate, metal-ligand interactions (such asnickel-histidine) may be cleaved by changing pH or introducing competingligands. The terms “cleave” or “cleaving” are also used herein to referto processes for separating linked assay components that do not requirebreaking covalent bonds, e.g., there are many well-establishedreversible binding pairs and conditions that may be employed (includingthose that have been identified in the art of affinity chromatography).By way of example, the binding of many antibody-ligand pairs can bereversed through changes in pH, addition of protein denaturants orchaotropic agents, addition of competing ligands, etc.

The targeting agents may be pairs of oligonucleotides comprisingcomplementary sequences. The preferred length is approximately 5 to 100bases, preferably, approximately, 10 to 50 bases, and more preferablyapproximately 10 to 2.5 bases. In addition, the targetingoligonucleotides sequences need not be identical in length and incertain embodiments it may be beneficial to provide one targetingoligonucleotide sequence that is longer than its binding partner, e.g.,by up to 25 bases, or up to 15 bases, or up to 10 bases. Known methodsthat are commonly employed for strand separation employ i) temperaturesabove the melting temperature for the complex, ii) use an alkaline pH of11 (or higher) or a low pH; iii) use high ionic strength and/or iv) usenucleic acid denaturants such as formamide. Other methods for strandseparation include the use of helicase enzymes such as Rep protein ofE.coli that can catalyse the unwinding of the DNA, or binding proteinssuch as 32-protein of E. coli phage T4 that act to stabilize thesingle-stranded form of DNA. In specific embodiments, dissociation ofcomplementary nucleic acid strands is accomplished by exposing thestrands to elevated temperature greater than 60° C.

The methods of the present invention may be used in a variety of assaydevices and/or formats. The assay devices may include, e.g., assaymodules, such as assay plates, cartridges, multi-well assay plates,reaction vessels, test tubes, cuvettes, flow cells, assay chips, lateralflow devices, etc., having assay reagents (which may include targetingagents or other binding reagents) added as the assay progresses orpre-loaded in the wells, chambers, or assay regions of the assay module.These devices may employ a variety of assay formats for specific bindingassays, e.g., immunoassay or immunochromatographic assays. Illustrativeassay devices and formats are described herein below. In certainembodiments, the methods of the present invention may employ assayreagents that are stored in a dry state and the assay devices/kits mayfurther comprise or be supplied with desiccant materials for maintainingthe assay reagents in a dry state. The assay devices preloaded with theassay reagents can greatly improve the speed and reduce the complexityof assay measurements while maintaining excellent stability duringstorage. The dried assay reagents may be any assay reagent that can bedried and then reconstituted prior to use in an assay. These include,but are not limited to, binding reagents useful in binding assays,enzymes, enzyme substrates, indicator dyes and other reactive compoundsthat may be used to detect an analyte of interest. The assay reagentsmay also include substances that are not directly involved in themechanism of detection but play an auxiliary role in an assay including,but not limited to, blocking agents, stabilizing agents, detergents,salts, pH buffers, preservatives, etc. Reagents may be present in freeform or supported on solid phases including the surfaces of compartments(e.g., chambers, channels, flow cells, wells, etc.) in the assay modulesor the surfaces of colloids, beads, or other particulate supports.

In one embodiment, assay reagents may be provided in an assay devicethat includes one or more regions or zones used for reagent storage.These storage zones may include the reagent bound to a surface withinthe storage zone, such that the reagent is confined within that zoneuntil it is subjected to conditions sufficient to release the reagentfor use elsewhere in the device. For example, the storage zone mayinclude a surface-reagent complex comprising a reagent linked to a firsttargeting agent and a surface linked to a second targeting agent,wherein the reagent and the surface are linked in the surface-reagentcomplex, via a releasable binding interaction between the first andsecond targeting agents. In this embodiment, the reagent is releasedfrom the surface-reagent complex and the storage zone by subjecting thestorage zone to conditions sufficient to disrupt the releasable bindinginteraction between the first and second targeting agents. As describedherein, those conditions may include but are not limited to, subjectingthe storage zone to increased or decreased temperature, light, alteringthe pH of that zone, applying an electrical potential, changes in ionicstrength, adding a competitor, and combinations thereof.

The surface to which the second targeting agent, and thereby, thereagent, is linked, may be any solid support that can be incorporatedwithin or confined to the storage zone. For example, the surface may bethe surface of one or more particles, as described herein, present inthe storage zone. Alternatively, the surface is a surface of the storagezone, for example, a surface of a compartment, channel, conduit, well,etc., within the storage zone. Preferably, the storage zone surface isroughened or includes one or more raised features or indentations thatincrease the relative surface area within the storage zone available tohold surface-reagent complexes. In one embodiment, the storage zonesurface includes surface area-enhancing features that increase thesurface area, such that the surface area accessible to a componentcapable of binding to that surface is at least two-fold larger than thesurface area of a flat surface. In a preferred embodiment, the surfacearea accessible for binding is at least three-fold larger than thesurface area of a flat surface. The high surface area support can beprovided by roughening a surface or otherwise providing threedimensional texture to a surface. A variety of established approachesfor preparing roughened or textured surfaces will be available to oneskilled in the art. Included in these approaches is the production ofsurfaces with high aspect ratio features such as arrays of columns thatare prepared through conventional machining, micro-machining orlithography (e.g., approaches using LIGA or other micro-fabricationtechnologies as described in U.S. Pat. Nos. 5,707,799 and 5,952,173) orinjection molding.

The storage zone surface may also include a composite materialcomprising exposed particles distributed in a matrix. The compositematerial may include, but is not limited to, carbon particles, graphiticparticles, or carbon nanotubes, Optionally, the composite may be etched(e.g., by chemical or plasma etching) to expose more particles andincrease the surface roughness. In one specific example, the surface isprovided by a printed carbon ink. In another embodiment, the storagezone surface may include a porous support that provides an enhancedsurface area through the surface area available in its pores. Suchporous supports include porous membranes (such as filtration membranesand lateral flow membranes) and gels. Preferred gels include hydrogels.A number of suitable hydrogels are well established as supports forreagents, as are chemistries for linking reagents to hydrogels, forapplications such as affinity chromatography, solid phase synthesis ofbiological polymers and binding assays, in applications. Examples ofsuch hydrogels include, but are not limited to, polymers of sugars(polysaccharides), acrylic acid, acrylates, acrylamides, ethyleneglycol, propylene glycol. The hydrogels may be cross-linked and/or maybe co-polymers of different monomer components.

An assay device that incorporates a storage zone for reagents alsoincludes a use zone configured to use those reagents in an assayconducted in that device. Therefore, once the reagent is released fromthe surface-reagent complex, free reagent is available for use in anassay conducted in the use zone. Free reagent is transferred from thestorage zone to the use zone, wherein it can participate in an assay foran analyte of interest. That assay may involve one or more additionalreagents present in the use zone or otherwise supplied to the use zone.In one embodiment, the use zone may include one or more additionalreagents bound to a solid support within the use zone and/or dried on asurface of the use zone. In a specific embodiment, the reagent is abinding reagent capable of binding an analyte of interest in a sample,and the use zone includes an additional ti reagent, bound to a solidsupport within the use zone, wherein that additional reagent also bindsthe analyte of interest. In this embodiment, the analyte present in thesample binds to the surface of the use zone via the additional reagent,as well as to the free reagent transferred from the storage zone to forma sandwich complex. The binding reagent may include a detectable label,e.g., an ECL label, and the analyte may be detected in the use zone bydetecting the presence or absence of the label, e.g., via measuringelectrochemiluminescence emitted in the use zone. The sample may beintroduced to the use zone directly or the sample is first introduced tothe storage zone and thereafter, the sample flows from the storage zoneto the use zone. The reagent may be released prior to contacting thestorage zone with sample or after the storage zone is contacted withsample. In one embodiment, sample is introduced to the storage zone,which is then subjected to conditions required for release of thereagent from the surface-reagent complex. Thereafter, the sample and thefree reagent are optionally incubated prior to transferring thesample-reagent mixture to the use zone.

In a preferred embodiment, the storage zone and the use zone are influidic communication along a fluid path. For example, the assay devicemay be a cartridge and the storage zone and the use zone are positionedwithin the cartridge along a fluid path. Examples of this embodiment areshown in FIG. 7(a)-(c). In FIG. 7(a), the assay device includes astorage zone and at least two use zones and each of the storage zonesand use zones are in fluid communication. The use zones may beconfigured in the assay device in series, as shown in FIG. 7(a) or inparallel, as shown in FIG. 7(b). FIG. 7(c) shows yet anotherconfiguration of an assay device including multiple storage and usezones. In the embodiment shown in FIG. 7(c), the storage and use zonesare also in fluidic communication with sample and/or reagentcompartments within the assay device.

Another embodiment is shown in FIGS. 8(a)-8(f). The assay device ofFIGS. 8(a)-8(f) includes a storage zone including a firstsurface-reagent complex and a second surface-reagent complex and atleast two use zones, wherein the storage zone and the use zone are influidic communication via a fluidic network. Sample is introduced into acompartment of the device in panel (a) and the fluidic network carriesthat sample to the use zones, as shown in panel (b). Panels (b) and (c)also shows that diluents can be passed through the storage zone (underconditions that do not release the surface-reagent complexes) andcarried through the fluidic network to the use zones to provide anoptional wash of the use zones. Diluent is then passed through thestorage zone while subjecting the storage zone to a condition thatreleases the first reagent, which is then carried to the fluidic networkin to use zone 1, as shown in panel (d). The second reagent is thenreleased by a second set of conditions and carried, via flow of diluentsthrough the microfluidic network, to use zone 2, as shown in panel (e).Finally, the use zones are optionally washed to remove excess reagent,as shown in panel (f).

In one embodiment, the storage zone and use zones are included within afluidic network further comprising one or more vent ports in fluidiccommunication with the storage and use zones (directly or through ventconduits) so as to allow the equilibration of fluid in the zones withthe atmosphere or to allow for the directed movement of fluid into orout of a specified zone by selectively sealing, opening (to atmosphericpressure) or applying positive or negative pressure to specific ventports.

In another embodiment, the assay device is a multi-well assay plate andthe use zone is positioned within a well of the plate, while the storagezone is located on a supplemental surface of the well that does notoverlap with the use zone.

In a further embodiment, the assay device may include one or moresurface-reagent complexes in the storage zone. In the embodimentdepicted in FIG. 8, for example, the storage zone includes a firstsurface-reagent complex (as described above) and also includes a secondsurface-reagent complex confined to the storage zone, the secondsurface-reagent complex including (i) a second reagent linked to a thirdtargeting agent; and (ii) a second surface linked to a fourth targetingagent, wherein the second reagent and the second surface are linked, inthe second surface-reagent complex, via a second releasable bindinginteraction between the third and fourth targeting agents; and the usezone is further configured to use the second reagent in the assay. Thevarious reagents stored within the storage zone may be used in one ormore assays conducted in the use zone, or each of the reagents storedwithin the storage zone may be used in each of the assays conducted inthe use zone. The reagents stored within the storage zone may beselectively released, one of the reagents may be released from thesurface-reagent complex composition by a first set of conditions thatdiffer from a second set of conditions used to release another reagentstored in the storage zone.

Additionally, the use zone may include two or more assay regions eachconfigured to use the reagents stored within the storage zone in one ormore assays conducted with a single sample in the device. In oneembodiment, the use zone includes a first assay region configured toconduct an assay for a first analyte of interest in a sample and the usezone may also include an additional assay region configured to conductan assay for an additional analyte of interest that may also be presentin the sample. Alternatively, the first assay region in the use zone maybe used to conduct a first assay for an analyte, while another assayregion in the use zone may be used to conduct a second assay for thesame analyte. Still further, the assay device may include a plurality ofuse zones each configured to use the reagents stored within the storagezone in one or more assays conducted with a single sample in the device.Each use zone may include one or more assay regions as described above.Moreover, the assay device may include a plurality of storage zones,e.g., for each use zone there is a corresponding storage zone. Variousconfigurations of an assay device including multiple use zones and/orstorage zones are shown in FIG. 7(a)-(c) and FIGS. 8(a)-8(f).

As described above, a storage zone may include a plurality of differentreagents as surface-reagent complexes. In one embodiment differentreagents are held in the storage zone by different releasable bindingreactions that are cleaved under different conditions. Therefore, bysubjecting each defined region of the storage zone to the appropriateconditions, each reagent is selectively released from the storage zone.The different reagents may be in surface-reagent complexes that areinter-mixed or held in distinct regions of the storage zone. Asdescribed herein, those conditions may include but are not limited to,subjecting the region to increased or decreased temperature, light,altering the pH of that region, changing the ionic strength, applying anelectrical potential, adding a competitor, and combinations thereof. Byusing binding reactions cleaved under different conditions, it ispossible to selectively release one reagent at a time fromsurface-reagent complexes in the storage zone. For example, one reagentmay be selectively released by heating while another may be selectivereleased by changing pH or one reagent may be selectively released usinga first competitor while another may be selectively released using asecond competitor. In another embodiment, different reagents may bereleased one a time using different releasable binding reactions thatrequire increasingly stringent cleavage conditions (such as increasingtemperature, increasing or decreasing pH, increasing competitorconcentration, increasing levels of light, increasing or decreasingionic strength, etc.). For example, a first reagent may be released at afirst temperature level and a second reagent may be subsequentlyreleased at a second higher temperature level.

In another embodiment, the storage zone may include a plurality ofdefined spatial regions, at least two of the different regions holdingdifferent reagents in surface-reagent complexes that hold the reagentsthrough releasable binding interactions as described above. In thisembodiment, cleavage of a reagent in a specific spatial region can becarried out by applying cleavage conditions (such as applying light,temperature, electrical potential, etc.) in a manner that confines thecleavage condition to the specific spatial region of interest. In thisembodiment, releasable binding interactions used for holding differentreagents can be the same or different, because release of individualreagents can be directed by application of the cleavage condition todefined region. In a preferred embodiment, the device is configured fora multiplexed assay measurement and the device includes (a) a storagezone comprising a surface-reagent complex confined to the storage zone,the surface-reagent complex including (i) a reagent linked to a firsttargeting agent; and (ii) a surface linked to a second targeting agent,wherein the reagent and the surface are linked, in the surface-reagentcomplex, via a releasable binding interaction between the first andsecond targeting agents; and (b) a use zone comprising a plurality ofassay regions configured to use the reagent in a multiplexed assay for aplurality of analytes in a sample. The storage zone may further comprisea second surface-reagent complex confined to the storage zone, thesecond surface-reagent complex including (iii) a second reagent linkedto a third targeting agent; and (iv) a second surface linked to a fourthtargeting agent, wherein the second reagent and the second surface arelinked, in the second surface-reagent complex, via a second releasablebinding interaction between the third and fourth targeting agents; andthe use zone is ti further configured to use the second reagent in themultiplexed assay. In this regard, the use zone comprises two or moreassay regions each configured to use the reagent and the second reagentin one or more assays conducted with the sample in the assay device, andthis configuration of assay device enables the conduct of a plurality ofassays in the use zone with the reagent and optionally, a secondreagent. The use zone may include a first assay region configured toconduct an assay for a first analyte of interest in the sample and anadditional assay region configured to conduct an assay for an additionalanalyte of interest in the sample, and an assay in such a devicecomprises the following steps:

(x) introducing the sample to the use zone via the storage zone;

(y) introducing a diluent to the storage zone;

(z) subjecting the storage zone to a condition that releases the reagentfrom the surface-reagent complex;

(xx) transferring the reagent from the storage zone to the first assayregion and the second assay region; and

(yy) conducting the assays in the first and second assay regions,respectively.

The conducting step of each assay may be performed simultaneously orsequentially.

Alternatively, an assay method may include an incubation step betweenthe sample and free reagent before the mixture of sample and freereagent is introduced to the use zone: Such a method would include thefollowing steps:

(x) introducing the sample to the storage zone;

(y) subjecting the storage zone to a condition that releases the reagentfrom the surface-reagent complex, and optionally incubated the samplewith the free reagent in the storage zone;

(z) transferring the mixture formed in (y) from the storage zone to thefirst assay region and the second assay region; and

(xx) conducting the assays in the first and second assay regions,respectively.

Still further, the use zone may include a first assay region configuredto conduct an assay for a first analyte of interest in the sample and anadditional assay region in the use zone configured to conduct an assayfor an additional analyte of interest in the sample, and an assay insuch a device may comprise:

(x) introducing the sample to the use zone via the storage zone;

(y) introducing a diluent to the storage zone and

-   -   i) subjecting the storage zone to a condition that releases the        reagent from the surface-reagent complex;    -   ii) subjecting the storage zone to an additional condition that        releases a second reagent from a second surface-reagent complex;

(z) transferring the reagent and the second reagent from the storagezone to the first and second assay regions; and

(xx) conducting the assays in the first and second assay regions.

The conducting step of each assay may be performed simultaneously orsequentially. Likewise, the transfer of the reagent and the secondreagent may be done simultaneously or sequentially.

Alternatively, an assay method using a device that includes a firstassay region configured to conduct an assay for a first analyte ofinterest in the sample and an additional assay region in the use zoneconfigured to conduct an assay for an additional analyte of interest inthe sample may also include an incubation step, i.e.,

(x) introducing the sample to the storage zone;

-   -   i) subjecting the storage zone to a condition that releases the        reagent from the surface-reagent complex;    -   ii) subjecting the storage zone to an additional condition that        releases a second reagent from a second surface-reagent complex;    -   (iii) incubating the storage zone with the free reagent and free        second reagent formed in steps (x)(i) and (x)(ii);

(y) transferring the mixture formed in step (x)(iii) from the storagezone to the first and second assay regions; and

(z) conducting the assays in the first and second assay regions.

In one specific embodiment, the assay device is a cartridge, such asthat described in application Ser. No. 61/284,276, filed Dec. 16, 2009,the disclosure of which is incorporated herein by reference. As shown,e.g., in FIG. 9 of U.S. Ser. No. 61/284,276, a cartridge may includevarious compartments, i.e., a sample chamber, an assay reagent chamber,waste chambers, and detection chambers, as well as a fluidic networkthat connects various compartments and/or fluid ports/vents. The storagezone may be incorporated within, e.g., a reagent chamber, and likewise,the use zone may be included within, e.g., the detection chamber.Additionally or alternatively, an additional storage chamber may beincorporated within the cartridge described therein.

In another specific embodiment, the assay device is a multi-well assayplate, such as that described in co-pending application Ser. No.11/642,970, filed Dec. 21, 2006, now U.S. Pat. No. 7,897,448, thedisclosure of which is incorporated herein by reference. The assay platemay include a plate body with a plurality of wells defined therein,wherein the plurality of wells includes a binding surface having acapture reagent immobilized therein, and an additional reagent locatedon a surface of the plate or well that does not overlap with the bindingsurface. In one embodiment, the additional reagent is located on areagent storage shelf positioned on a wall of a well. Alternatively, anassay plate may include assay wells that are connected to dedicatedreagent spaces located in the regions between the assay wells. In suchan embodiment, a reagent space may be in fluidic communication with thesurrounding wells via e.g., a notch. In addition, suitable assay platesare described in U.S. patent application Ser. No. 11/642,970, now U.S.Pat. No. 7,897,448, the disclosure of which is incorporated herein byreference.

(iii) Solid Phases

A wide variety of solid phases are suitable for use in the methods ofthe present invention including conventional solid phases from the artof binding assays. Solid phases may be made from a variety of differentmaterials including polymers (e.g., polystyrene and polypropylene),ceramics, glass, composite materials (e.g., carbon-polymer compositessuch as carbon-based inks). Suitable solid phases include the surfacesof macroscopic objects such as an interior surface of an assay container(e.g., test tubes, cuvettes, flow cells, cartridges, wells in amulti-well plate, etc.), slides, assay chips (such as those used in geneor protein chip measurements), pins or probes, beads, ti filtrationmedia, lateral flow media (for example, filtration membranes used inlateral flow test strips), etc.

Suitable solid phases also include particles (including but not limitedto colloids or beads) commonly used in other types of particle-basedassays e.g., magnetic, polypropylene, and latex particles, materialstypically used in solid-phase synthesis e.g., polystyrene andpolyacrylamide particles, and materials typically used inchromatographic applications e.g., silica, alumina, polyacrylamide,polystyrene. The materials may also be a fiber such as a carbon fibril.Microparticles may be inanimate or alternatively, may include animatebiological entities such as cells, viruses, bacterium and the like.

The particles used in the present method may be comprised of anymaterial suitable for attachment to one or more binding partners and/orlabels, and that may be collected via, e.g., centrifugation, gravity,filtration or magnetic collection. A wide variety of different types ofparticles that may be attached to binding reagents are sold commerciallyfor use in binding assays. These include non-magnetic particles as wellas particles comprising magnetizable materials which allow the particlesto be collected with a magnetic field. In one embodiment, the particlesare comprised of a conductive and/or semiconductive material, e.g.,colloidal gold particles.

The microparticles may have a wide variety of sizes and shapes. By wayof example and not limitation, microparticles may be between 5nanometers and 100 micrometers. Preferably microparticles have sizesbetween 20 nm and 10 micrometers. The particles may be spherical,oblong, rod-like, etc., or they may be irregular in shape.

The particles used in the present method may be coded to allow for theidentification of specific particles or subpopulations of particles in amixture of particles. The use of such coded particles has been used toenable multiplexing of assays employing particles as solid phasesupports for binding assays. In one approach, particles are manufacturedto include one or more fluorescent dyes and specific populations ofparticles are identified based on the intensity and/or relativeintensity of fluorescence emissions at one or more wave lengths. Thisapproach has been used in the Luminex xMAP systems (see, e.g., U.S. Pat.No. 6,939,720) and the Becton Dickinson Cytometric Bead Array systems.Alternatively, particles may be coded through differences in otherphysical properties such as size, shape, imbedded optical patterns andthe like.

In certain embodiments of assays of the invention, particles may have adual role as both i) a solid phase support used in an analyteconcentration, collection and/or separation step and ii) as a detectablelabel or platform for detectable labels in a measurement step. In oneexample, a method of conducting a binding assay may comprise contactinga sample comprising an analyte with a particle linked to a first bindingreagent that binds that analyte to form a complex comprising the analytebound to the first binding reagent. The complex is then collected bycollection of the particle (via magnetic collection, centrifugation,gravity sedimentation, etc.) and some or all of the unbound componentsof the sample are separated from the complex by removing some or all ofthe sample volume and, optionally, washing the collected particles. Thecomplex is then released by resuspending the particles in the originalor a new liquid media. The complex on the particle is then contactedwith a second binding reagent bound to a solid phase, the second bindingreagent binding the complex so as to bring the complex and particle to asurface of the solid phase. The amount of analyte in the sample ismeasured by measuring the amount of analyte bound to the solid phase,which in turn is measured by measuring the amount of particles bound tothe solid phase (either by directly measuring the particles or bymeasuring detectable labels in or on the particles by, e.g., themeasurement approaches described below).

The invention also includes assay methods that employ magnetic particlesas detectable labels or as platforms for detectable labels in a bindingassay. Advantageously, when using magnetic particles as a label or alabel platform, a magnetic field can be applied to speed the kineticsfor the binding of i) assay components linked to a magnetic particle toii) binding reagents immobilized on a solid phase.

Accordingly, one embodiment is a method for conducting a binding assaycomprising

(a) contacting (i) a sample comprising a target analyte with (ii) amagnetic particle linked to a first binding reagent that binds thetarget analyte and thereby forms a complex comprising the target analytebound to the first binding reagent;

(b) contacting a solution comprising the complex with a second bindingreagent bound to a solid phase, wherein the second binding reagent bindsto the complex;

(c) applying a magnetic field to concentrate the particles near to thesolid phase and thereby accelerating the rate of binding between thecomplex and the second binding reagent and

(d) measuring the amount of the analyte bound to the solid phase.

Optionally, such a method may also include, prior to step (b),collection and release steps as described elsewhere in this applicationso as to pre-concentrate the analyte and/or remove interferents from thesample. The magnetic particles used in such method are, preferably,between 10 nm and 10 um in diameter, more preferably between 50 nm and 1um. The step of applying a magnetic field may be achieved through theuse of permanent or electromagnets, e.g., by placing the magnet on theopposite side of the solid phase relative to the second binding reagent,Optionally, the magnet or magnetic field is translated and/or rotatedalong the solid phase so as to move the particles along the bindingsurface and allow the particles to interrogate the surface for availablebinding sites. Alternatively, or in conjunction with movement of themagnet/field, the magnetic field is intermittently removed and, whilethe magnetic field is removed, the particles are resuspended (e.g., bymixing) and then reconcentrated on the solid phase (thereby, allowingfor allowing the particles to change rotational orientation on thesurface and allowing them to interrogate additional areas on thesurface. The method may also include a washing step, prior to themeasuring step, to remove unbound particles. During such a washing step,the magnetic field is removed to allow for non-bound particles to bewashed away. Alternatively, a magnetic field above the surface can beused to pull unbound particles away from the surface. The magneticreaction acceleration approach may also be applied to multiplexed assaymethods, as described elsewhere in this application, e.g., the solidphase may include an array of a plurality of different second bindingreagents for use in array-based multiplexed measurements.

(iv) Collection and Release

Collection, as used herein, refers to the physical localization of amaterial in a ti mixture. Collection includes the localization of amaterial through binding reactions or adsorption. For example, amaterial in a mixture may be collected on a solid phase by adsorption ofthe material on the solid phase or by binding of the material to bindingreagents on the solid phase. Collection is not, however, limited tolocalization at a solid phase and may also include techniques in the artfor localizing materials at a location/volume within a larger fluidvolume, for example, localization of materials through the use ofoptical tweezers (which use light to manipulate microscopic objects assmall as a single atom, wherein the radiation pressure from a focusedlaser beam is able to trap small particles), electric or magneticfields, focused flow, density gradient centrifugation, etc.

Certain embodiments of the invention include the collection ofmicroparticles or materials that are bound to microparticles. Suitablecollection methods include the many methods known in the art ofmicroparticle-based assays that achieve localization of microparticlesfrom a suspension. These include sedimentation under gravity or bycentrifugation, filtration onto a filter or porous membrane,localization (of magnetizable particles) by application of a magneticfield, binding or adsorption of the particles to a macroscopic solidphase, use of optical tweezers, etc.

Release, as used herein, refers to delocalization of a previouslycollected material. Materials that are held at a localized positionthrough chemical bonds or through specific or non-specific bindinginteractions may be allowed to delocalize by breaking the bond orinteraction so that the materials may diffuse or mix into thesurrounding media. There are many well-established cleavable chemicallinkers that may be used that provide a covalent bond that may becleaved without requiring harsh conditions. For example, disulfidecontaining linkers may be cleaved using thiols or other reducing agents,cis-diol containing linkers may be cleaved using periodate, metal-ligandinteractions (such as nickel-histidine) may be cleaved by changing pH orintroducing competing ligands. Similarly, there are manywell-established reversible binding pairs that may be employed(including those that have been identified in the art of affinitychromatography). By way of example, the binding of many antibody-ligandpairs can be reversed through changes in pH, addition of proteindenaturants or chaotropic agents, addition of competing ti ligands, etc.Other suitable reversible binding pairs include complementary nucleicacid sequences, the hybridization of which may be reversed under avariety of conditions including changing pH, increasing saltconcentration, increasing temperature above the melting temperature forthe pair and/or adding nucleic acid denaturants (such as formamide).Such reversible binding pairs may be used as targeting agents (asdescribed above), e.g., a first targeting agent may be linked to a firstbinding reagent that binds an analyte, a second targeting agent may belinked to a solid phase, and a binding interaction of the first andsecond targeting agents may be used to reversibly immobilize the firstbinding reagent on the solid phase.

Release also includes physical delocalization of materials by, forexample, mixing, shaking, vortexing, convective fluid flow, mixing byapplication of magnetic, electrical or optical forces and the like.Where microparticles or materials bound to microparticles have beencollected, such physical methods may be used to resuspend the particlesin a surrounding matrix. Release may simply be the reverse of a previouscollection step (e.g., by any of the mechanisms described above) orcollection and release could proceed by two different mechanisms. In onesuch example, collection of materials (such as an analyte or a complexcomprising an analyte) bound to a particle can be achieved by physicalcollection of the particle. The materials are then released by cleavinga bond or reversing a binding reaction holding the material on theparticle. In a second such example, materials (such as an analyte of acomplex comprising an analyte are collected on a surface through abinding interaction with a binding reagent that is linked to thesurface. The material is then released by breaking a bond or a secondbinding interaction linking the binding reagent to the surface.

Collection followed by release may be used to concentrate and/or purifyanalytes in a sample. By collecting in a first volume and releasing intoa second smaller volume, an analyte in a sample may be concentrated.Through concentration, it is often possible to significantly improve thesensitivity of a subsequent measurement step. By collecting from asample and removing some or all of the uncollected sample, potentialassay interferents in the sample may be reduced or eliminated.Optionally, removal of the unbound sample may include washing acollected material with and releasing the collected material intodefined liquid reagents (e.g., assay or wash buffers) so as to provide auniform matrix for subsequent assay steps.

(iv) Measurement Methods

The methods of the invention can be used with a variety of methods formeasuring the amount of an analyte and, in particular, measuring theamount of an analyte bound to a solid phase. Techniques that may be usedinclude, but are not limited to, techniques known in the art such ascell culture-based assays, binding assays (including agglutinationtests, immunoassays, nucleic acid hybridization assays, etc.), enzymaticassays, colorometric assays, etc. Other suitable techniques will bereadily apparent to one of average skill in the art. Some measurementtechniques allow for measurements to be made by visual inspection,others may require or benefit from the use of an instrument to conductthe measurement.

Methods for measuring the amount of an analyte include label freetechniques, which include but are not limited to i) techniques thatmeasure changes in mass or refractive index at a surface after bindingof an analyte to a surface (e.g., surface acoustic wave techniques,surface plasmon resonance sensors, ellipsometric techniques, etc.), ii)mass spectrometric techniques (including techniques like MALDI, SEMI,etc. that can measure analytes on a surface), iii) chromatographic orelectrophoretic techniques, iv) fluorescence techniques (which may bebased on the inherent fluorescence of an analyte), etc.

Methods for measuring the amount of an analyte also include techniquesthat measure analytes through the detection of labels which may beattached directly or indirectly (e.g., through the use of labeledbinding partners of an analyte) to an analyte. Suitable labels includelabels that can be directly visualized (e.g., particles that may be seenvisually and labels that generate an measurable signal such as lightscattering, optical absorbance, fluorescence, chemiluminescence,electrochemiluminescence, radioactivity, magnetic fields, etc). Labelsthat may be used also include enzymes or other chemically reactivespecies that have a chemical activity that leads to a measurable signalsuch as light scattering, absorbance, fluorescence, etc. The use ofenzymes as labels has been well established in in Enzyme-LinkedImmunoSorbent Assays, also called ELISAs, Enzyme ImmunoAssays or EIAs.In the ELISA format, an unknown amount of antigen is affixed to asurface and then a specific antibody is washed over the surface so thatit can bind to the antigen. This antibody is linked to an enzyme, and inthe final step a substance is added that the enzyme converts to aproduct that provides a change in a detectable signal. The formation ofproduct may be detectable, e.g., due a difference, relative to thesubstrate, in a measurable property such as absorbance, fluorescence,chemiluminescence, light scattering, etc. Certain (but not all)measurement methods that may be used with solid phase binding methodsaccording to the invention may benefit from or require a wash step toremove unbound components (e.g., labels) from the solid phase.Accordingly, the methods of the invention may comprise such a wash step.

In one embodiment, an analyte(s) of interest in the sample may bemeasured using electrochemiluminescence-based assay formats, e.g.electrochemiluminescence (ECL) based immunoassays. The high sensitivity,broad dynamic range and selectivity of ECL are important factors formedical diagnostics. Commercially available ECL instruments havedemonstrated exceptional performance and they have become widely usedfor reasons including their excellent sensitivity, dynamic range,precision, and tolerance of complex sample matrices. Species that can beinduced to emit ECL (ECL-active species) have been used as ECL, labels,e.g., i) organometallic compounds where the metal is from, for example,the noble metals of group VIII, including Ru-containing andOs-containing organometallic compounds such as thetris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and relatedcompounds. Species that participate with the ECL label in the ECLprocess are referred to herein as ECL coreactants. Commonly usedcoreactants include tertiary amines (e.g., see U.S. Pat. No. 5,846,485),oxalate, and persulfate for ECL from RuBpy and hydrogen peroxide for ECLfrom luminol (see, e.g., U.S. Pat. No. 5,240,863). The light generatedby ECL labels can be used as a reporter signal in diagnostic procedures(Bard et al., U.S. Pat. No. 5,238,808, herein incorporated byreference). For instance, an ECL label can be covalently coupled to abinding agent such as an antibody, nucleic acid probe, receptor orligand; the participation of the binding reagent in a bindinginteraction can be monitored by ti measuring ECL, emitted from the ECLlabel. Alternatively, the ECL signal from an ECL-active compound may beindicative of the chemical environment (see, e.g., U.S. Pat. No.5,641,623 which describes ECL assays that monitor the formation ordestruction of ECL coreactants). For more background on ECL, ECL labels,ECL assays and instrumentation for conducting ECL assays see U.S. Pat.Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623;5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141;5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863;6,207,369; 6,214,552 and 5,589,136 and Published PCT Nos. WO 99/63347;WO 00/03233; WO 99/58962; WO 99/32662; WO 99/14599; WO 98/12539; WO97/36931 and WO 98/57154, all of which are incorporated herein byreference.

The capture/collection and release methods of the invention may beapplied to singleplex or multiplex formats where multiple assaymeasurements are performed on a single sample. Multiplex measurementsthat can be used with the invention include, but are not limited to,multiplex measurements i) that involve the use of multiple sensors; ii)that use discrete assay domains on a surface (e.g., an array) that aredistinguishable based on location on the surface; iii) that involve theuse of reagents coated on particles that are distinguishable based on aparticle property such as size, shape, color, etc.; iv) that produceassay signals that are distinguishable based on optical properties(e.g., absorbance or emission spectrum) or v) that are based on temporalproperties of assay signal (e.g., time, frequency or phase of a signal).

(v) Assay Formats

One embodiment of the present invention employs a specific bindingassay, e.g., an immunoassay, immunochromatographic assay or other assaythat uses a binding reagent. The immunoassay or specific binding assayaccording to one embodiment of the invention can involve a number offormats available in the art. The antibodies and/or specific bindingpartners can be labeled with a label or immobilized on a surface. Thus,in one embodiment, the detection method is a binding assay, e.g., animmunoassay, receptor-ligand binding assay or hybridization assay, andthe detection is performed by contacting an assay composition with oneor more detection molecules capable of specifically binding with ananalyte(s) of interest in the sample.

In one embodiment, the assay uses a direct binding assay format. Ananalyte is bound to a binding partner of the analyte, which may beimmobilized on a solid phase. The bound analyte is measured by directdetection of the analyte or through a label attached to the analyte(e.g., by the measurements described above).

In one embodiment, the assay uses a sandwich or competitive bindingassay format. Examples of sandwich immunoassays performed on test stripsare described in U.S. Pat. No. 4,168,146 to Grubb et al. and U.S. Pat.No. 4,366,241 to Tom et al., both of which are incorporated herein byreference. Examples of competitive immunoassay devices suitable for usewith the present methods include those disclosed in U.S. Pat. No.4,235,601 to Deutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S.Pat. No. 5,208,535 to Buechler et al., all of which are incorporatedherein by reference.

In a sandwich assay, analyte in the sample is bound to a first bindingreagent and a second labeled binding reagent and the formation of this“sandwich” complex is measured. In a solid phase sandwich assay, thefirst binding reagent is immobilized on a solid phase and the amount oflabeled antibody on the solid phase, due to formation of the sandwichcomplex, is then measured. The signal generated in a sandwich assay willgenerally have a positive correlation with the concentration of theanalyte. Various configurations of sandwich assays that use the methodsof the present invention are shown in FIGS. 1-4. In one embodiment,e.g., in FIG. 1(a), the assay includes contacting a sample comprising atarget analyte with a particle or solid phase linked to a first bindingreagent that binds the target analyte, thereby forming a complexcomprising the target analyte bound to the first binding reagent. Thecomplex is collected, separated and released, as described herein, andthen a sandwich is formed by contacting the complex with an additionalbinding reagent (e.g., a second binding reagent). As shown in FIG. 1(a)and FIG. 1(b), the particle or solid phase may or may not be cleavedfrom the complex prior to contacting the complex with an additionalbinding reagent.

In a competitive assay, unlabelled analyte in the test sample ismeasured by its ability to compete with labeled or immobilized analyte.In the example of competitive assays employing labeled analytes, theunlabeled analyte in a sample blocks the ability of the labeled analyteto bind a binding reagent by occupying the binding site. Thus, in acompetitive assay, the signal generated has an inverse correlation withthe concentration of analyte in a sample. FIGS. 6(a) and 6(b) show theuse of the methods of the present invention in a two step competitiveformat. As in FIG. 1(a), the analyte of interest in the sample ispre-concentrated. Labeled analyte bound to a solid support is incubatedwith the pre-concentrated analyte complex. FIGS. 6(a) and 6(b) serve toillustrate how the methods of the present invention may be used in acompetitive assay format. The skilled artisan will understand thatalternate configurations of a competitive immunoassay may be achievedusing the methods of the present invention without undueexperimentation.

(vi) Specific Embodiments

In one embodiment, a method is provided for conducting a binding assaycomprising contacting a sample comprising a target analyte, A, and whichmay also contain various sample contaminants as shown in FIG. 1(a), witha particle linked to a first binding reagent that binds the targetanalyte and thereby forms a complex comprising the target analyte boundto the first binding reagent. Once the sample is mixed with the particleto form the complex, the complex is collected. This collection step mayinvolve accumulation of the complex at a surface, e.g., bycentrifugation of the particles, allowing the particles to rise orsettle under gravity, filtering the particles onto a filtration media,magnetically collecting the particles (in the case of magneticparticles), etc. Alternatively, the collection step may involveaccumulation of the complex within a defined volume within the sample,e.g., by holding the particles in this defined volume through the use ofoptical tweezers or focused flow. Optionally, the unbound components ofthe sample are then separated from the complex, e.g., by removing all orpart of the non-collected components and/or by washing the collectedcomplex with an additional assay medium or wash buffer. Thereafter, thecomplex is released, e.g., resuspended into the assay medium, and thecomplex is contacted with a second binding reagent bound to a solidphase, wherein the second binding reagent binds to the complex. Theamount of analyte is detected by measuring the amount of a detectablelabel linked to an assay component bound to the solid phase. Thedetectable label may be linked to the first binding reagent, an optionalthird binding reagent, if one is used in the assay format, the particleor an additional assay component that is comprised within or bound tothe complex.

A variety of approaches are provided for conducting the collection andrelease steps described above and for providing the labeled reagent.FIG. 1(a) shows a method with the following steps: (i) a first bindingreagent linked to a particle binds to the analyte to form a complex, (iiand iii) the complex is collected and released by collection andresuspension of the particle during which steps the analyte may beconcentrated and/or separated from contaminants in the sample, (iv) thecomplex binds to a second binding reagent on a solid phase and (v) thecomplex is contacted with a labeled third binding reagent that binds theanalyte in the complex such that it can be detected. FIG. 1(b) shows amethod similar to the one in FIG. 1(a), except that the complex isreleased in step (iii) by cleaving the first binding reagent from theparticle instead of simply resuspending the particle. FIGS. 1(c) and1(d) show methods similar to the one in FIG. 1(a) except that that thelabel is attached to (or incorporated within) the particle (FIG. 1(c))or attached to the first binding reagent (FIG. 1(d)) and the step ofcontacted the complex with a labeled third binding reagent is omitted.Alternatively, if the particle is measured directly (e.g., by directvisual observation of the particle), the label may be omitted. FIG. 1(e)shows a method similar to the one in FIG. 1(b) except that the label isattached to the first binding reagent and the step of contacting thecomplex with a labeled third binding reagent is omitted.

The measuring step may comprise any suitable method of measuring thepresence of a detectable label in a sample (see the Measurement Methodssection), e.g., optical absorbance, fluorescence, phosphorescence,chemiluminescence, light scattering or magnetism. In one embodiment, thedetectable label is an electrochemiluminescent label and the measuringstep comprises measuring an ECL signal and correlating that signal withan amount of analyte in the sample. Thus, the measuring step may furthercomprise contacting the complex with an electrode and applying a voltagewaveform to the electrode to generate ECL.

The methods described in FIGS. 1(a)-1(e) may be applied to multiplexmeasurements for multiple analytes in a sample. In such methods, thefirst, second and ti third binding reagents (if present) may be selectedto bind multiple analytes (e.g., the use of poly-dT as a binding reagentto capture multiple mRNAs in a sample through the common poly-dA tailsequence) or, alternatively, the methods may employ a plurality ofdifferent first binding reagents, second binding reagents and/or thirdbinding reagents to bind to the multiple analytes. To allow forindependent measurement of different analytes, such multiplex methodsemploys at least one of the group consisting of i) a plurality ofdifferent first binding reagents, ii) a plurality of second bindingreagents and iii) a plurality of third binding reagents (the differentreagents within (i), (ii) or (iii) being selected for their ability topreferentially bind a target analyte relative to other target analytes).Where a plurality of first binding reagents are used, individualparticles may be attached to mixtures of the different first bindingreagents or, alternatively, the particles may be prepared so thatindividual particles are attached to only one type of first bindingreagent (e.g., such that an individual particle preferentially binds oneof the target analytes relative to other target analytes).

The multiplex methods may use a variety of approaches for independentlymeasuring different analytes. In one embodiment, a plurality of labeledbinding reagents with different preferences for target analytes may beused (e.g., a plurality of different labeled third binding reagents asin FIGS. 1(a) and 1(b), a plurality of different labeled first bindingreagents as in FIG. 1(e) or a plurality of different labeled firstbinding reagent-particle conjugates as in FIGS. 1(c) and 1(d)). Thelabels on the different labeled reagents (or, alternatively, theparticles in the particle conjugates) are selected to providedistinguishable assay signals such that the different labeled reagentsand, therefore, the different target analytes, can be measuredindependently. In another embodiment, a plurality of second bindingreagents with different preferences for target analytes may be used. Thedifferent second binding reagents may be patterned into differentdiscrete binding domains on one or more solid phases (e.g., as in abinding array) such that assay signals generated on the differentbinding domains and, therefore, the different analytes, can be measuredindependently (e.g., by independently addressing binding domains onelectrode arrays or by independently measuring light emitted fromdifferent binding domains in a luminescence assay). Alternatively, thedifferent second binding reagents ti may be coupled to different codedbeads (as described in the Solid Phases section) to allow for thedifferent analytes to be measured independently.

In an alternative embodiment, a method of conducting a binding assay isprovided as shown in FIGS. 2(a)-2(b), which comprises contacting asample comprising a target analyte with a first solid phase, S, linkedto a first binding reagent that binds the target analyte and forms acomplex comprising the target analyte bound to the first bindingreagent. Once the sample is contacted with the first solid phase, theunbound components of the sample are separated from the complex, thecomplex is released from the solid phase into the assay medium and thefirst solid phase is removed from the first binding reagent. Thereafter,the released complex is contacted with a second solid phase comprising asecond binding reagent that binds to the complex, and the amount ofanalyte bound to the second solid phase is quantified. The detectablelabel may be linked to the first binding reagent, an optional thirdbinding reagent, if one is used in the assay format, the particle or anadditional assay component that is comprised within or bound to thecomplex. In FIG. 2(a), the label is attached to a third binding reagent(and the method includes the step of contacting the complex with thethird binding reagent), whereas the label is attached to the firstbinding reagent in FIG. 2(b).

As described for FIG. 1, the methods described in FIG. 2 may also beextended to multiplex measurements, e.g., by employing at least one ofthe group consisting of i) a plurality of different first bindingreagents, ii) a plurality of second binding reagents and iii) aplurality of third binding reagents (the different reagents within (i),(ii) or (iii) being selected for their ability to preferentially bind atarget analyte relative to other target analytes).

The invention also provides a method of conducting a multiplexed bindingassay for a plurality of analytes that includes contacting (i) a samplewith (ii) one or more first solid phases linked to one or more firstbinding reagents that bind the analytes to form complexes comprising theanalytes bound to the first binding reagents. The unbound components ofthe sample are, optionally, separated from the complexes. The complexesare released and then contacted with a plurality of binding domainscomprising second binding reagents that bind to the complexes, whereineach binding domain comprises a second binding reagent that binds to acomplex comprising a secondary target analyte. Thereafter, the amount ofanalyte bound to the binding domains is measured.

According to another embodiment, a multiplexed assay may comprise theacts of contacting at least a portion of a sample with one or morebinding surfaces comprising a plurality of binding domains, immobilizingone or more analytes on the domains and measuring the analytesimmobilized on the domains. In certain embodiments, at least two of thebinding domains differ in their specificity for analytes of interest. Inone example of such an embodiment, the binding domains are prepared byimmobilizing, on one or more surfaces, discrete domains of bindingreagents that bind analytes of interest. Optionally, the sample isexposed to a binding surface that comprises an array of bindingreagents. Optionally, the surface(s) may define, in part, one or moreboundaries of a container (e.g., a flow cell, well, cuvette, etc.) whichholds the sample or through which the sample is passed. The method mayalso comprise generating assay signals that are indicative of the amountof the analytes in the different binding domains, e.g., changes inoptical absorbance, changes in fluorescence, the generation ofchemiluminescence or electrochemiluminescence, changes in reflectivity,refractive index or light scattering, the accumulation or release ofdetectable labels from the domains, oxidation or reduction or redoxspecies, electrical currents or potentials, changes in magnetic fields,etc.

Assays of certain embodiments of the invention may employ targetingagents to link the target analyte with a binding reagent in the assaymedium. Such assay formats are illustrated in FIGS. 3(a)-3(e) and FIGS.4(a)-4(b), which are analogous to FIGS. 1(a)-1(e) and FIGS. 2(a)-2(b),except that the binding of analyte to a first binding reagent on a solidphase/particle takes place through two steps: (i(a)) contacting thefirst binding reagent linked to a first targeting agent to a particle(or other solid phase) linked to a second targeting agent that binds tothe first targeting agent (thus attaching the first binding reagent tothe particle or other solid phase) and (i(h)) contacting the firstbinding reagent with a sample comprising a target analyte that binds thefirst binding reagent. Step i(a) may occur before step i(b) (as shown inthe figures) or the two steps may occur in the reverse order orconcurrently. Steps i(a) and i(b) may both be carried out during theconduct of an assay or, alternatively, the first binding reagent may besupplied to the user ti pre-bound to the solid phase through thetargeting agents (e.g., if the targeting agents were pre-bound duringmanufacturing), in which case step i(a) may be omitted.

Thus, in one embodiment, the method includes contacting a samplecomprising a target analyte with a particle linked to a first bindingreagent that binds the target analyte, wherein the first binding reagentis linked to a first targeting agent and the particle is linked to asecond targeting agent, and the first binding reagent and the particleare linked via a binding reaction between the first and second targetingagents to form a complex comprising the target analyte bound to thefirst binding reagent (see e.g., FIG. 3(a)). The complex is thencollected and unbound components in the sample are separated from thecomplex. The complex is released and the released complex is contactedwith a second binding reagent bound to a solid phase, wherein the secondbinding reagent binds to the complex. The amount of analyte bound to thesolid phase is measured. As in the embodiments described above andillustrated in FIGS. 1(a)-1(e), the detectable label may be attached tovarious assay components in the medium, e.g., to a third bindingreagent, as in FIGS. 3(a)-3(b), to the particle, as in FIG. 3(c), or tothe first binding reagent, as in FIGS. 3(d)-3(e). Moreover, the complexis optionally cleaved from the particle prior to the detection step, asin FIGS. 3(b) and 3(d).

In one embodiment, the assay may include (a) contacting a samplecomprising a target analyte with a first solid phase linked to a firstbinding reagent that binds the target analyte, wherein the first bindingreagent is linked to a first targeting agent and the first solid phaseis linked to a second targeting agent, and the first binding reagent andthe first solid phase are linked via a binding reaction between thefirst and second targeting agents to form a complex comprising thetarget analyte bound to the first binding reagent (see e.g., FIGS.4(a)-4(b)). The complex is then collected and unbound components in thesample are separated from the complex. The complex is released, e.g.,resolubilized, and the first solid phase is removed. The releasedcomplex is contacted with a second binding reagent bound to a secondsolid phase, wherein the second binding reagent binds to the complex.The amount of analyte bound to the second solid phase is measured. Thedetectable label may be attached to any suitable assay component, e.g.,the first binding reagent, as in FIG. 4(b), or the third bindingreagent, as in FIG. 4(a).

The releasing step in the various assay formats described herein maycomprise cleaving a binding reagent from the particle (e.g., as shown inFIG. 1(b)). This may be accomplished by any suitable method, e.g.,subjecting the complex to increased temperature, pH changes, alteringthe ionic strength of the solution, competition, and combinationsthereof.

If a targeting agent is employed in the assay format, the releasing stepcomprises disassociating the first and second targeting agents, e.g., bysubjecting the complex to increased temperature, pH changes, alteringthe ionic strength of the solution, competition, and combinationsthereof as discussed above.

The measuring step in the various assay formats described herein maycomprise any suitable method of measuring the presence of a detectablelabel in a sample, e.g., optical absorbance, fluorescence,phosphorescence, chemiluminescence, light scattering or magnetism. Inone embodiment, the detectable label is an electrochemiluminescent labeland the measuring step comprises measuring an ECL signal and correlatingthat signal with an amount of analyte in the sample. Thus, the measuringstep may further comprise contacting the complex with an electrode andapplying a voltage waveform to the electrode to generate ECL.

By analogy to the description of FIGS. 1 and 2, the methods in FIGS. 3and 4 may also be extended to multiplex measurements, e.g., by employingat least one of the group consisting of i) a plurality of differentfirst binding reagents, ii) a plurality of second binding reagents andiii) a plurality of third binding reagents (the different reagentswithin (i), (ii) or (iii) being selected for their ability topreferentially bind a target analyte relative to other target analytes).In such multiplex methods, a common targeting reagent pair may be usedto link a plurality of different first binding reagents to thecorresponding particles or other solid phases. Alternatively, a uniquetargeting reagent pair may be used for each different first bindingreagent (e.g., a different set of complementary oligonucleotides may beused to target each of the different first binding reagents). Such anapproach may be used to i) target different first binding reagents todifferent distinguishable particles (e.g., particles bearingdistinguishable labels) or ii) enable multiplexing through the use of aplurality of different second binding reagents, each of which bindspreferentially to a different first targeting agent (thus preferentiallybinding complexes comprising one of the plurality of analytes)

EXAMPLES Example 1 Dual Use of Labeled Magnetic Particle to Concentrateand Detect Analytes of Interest

As shown in FIG. 5, magnetic particles are coated with antibodiesagainst the analytes of interest and a large number (e.g., greater than100) ECL labels. By attachment of the ECL labels to the antibodies(either before or after coating the antibodies on the particles), veryhigh numbers of labels can be easily achieved. A particle of only 60 nmin diameter can support roughly 160 antibody molecules, assuming about50 nm² of surface area per antibody. Thus, attachment of only 1 labelper antibody allows labeling ratios of greater than 100 labels perparticle to be achieved for 60 nm particles. Labeling ratios of greaterthan 1000 labels per particle are achieved by increasing the number oflabels per antibody and/or increasing the particle size).

A 1 mL or greater volume of sample is combined with the particles in acontainer and after incubating the mixture to allow the antibodies tobind their respective targets, a magnetic field is applied such that themagnetic particles collect on a surface in the container (a variety ofcommercial magnetic tube holders or probes are available for carryingout this step). The complexes are washed with buffered saline to removeunbound components of the sample. The magnetic field is removed and theparticles are then re-suspended in 100 uL of a suitable assay diluent,thus providing a 10-fold or greater increase in concentration relativeto the original sample. The particle-analyte complexes are transferredto an assay plate (e.g., a MULTI-ARRAY® 96-well assay plate, Meso ScaleDiagnostics, LLC, Gaithersburg, Md.) that includes a binding surfacecomprising an array of antibody binding reagents directed against theanalytes of interest. Complexes that bind the array are measured by ECLon a SECTOR® Imager instrument (Meso Scale Diagnostics, LLC). Themagnetic collection step provides for improvements in assay performanceby allowing for pre-concentration of analyte into a small volume andremoval of potential interferents in the sample.

Example 2 Assay Using Antibodies Coupled to Magnetic Particles ThroughOligonucleotide Hybridization Reactions

Magnetic particles are coated with oligonucleotides and a large number(greater than 100) ECL labels. Conjugates are formed comprisingantibodies against analytes of interest and oligonucleotidescomplementary to the oligonucleotides on the particles. The antibodyconjugates and particles are subjected to conditions sufficient tohybridize the complementary oligonucleotide sequences (e.g., appropriatetemperature, ionic strength and denaturing conditions, as describedhereinabove) and thereby coat the antibodies on the particles. Theseparticles are then used to assay for analytes of interest as describedin Example 1.

Example 3 Demonstration of the Release of Antibodies Coupled to MagneticParticles Through Oligonucleotide Hybridization Reactions

Magnetic beads (Dynalbeads® MyOne™-Streptavidin C1 beads, InvitrogenCorporation) were coated with a biotinylated oligonucleotide by thefollowing procedure: The beads (3 mg) were washed three times at 60° C.in hybridization buffer (20 mM Tris, 1 mM EDTA, 250 mM NaCl, 0.01%Triton-X at pH=8 and 0.1% BSA). The beads were then coated at roomtemperature with 750 pmoles of a 19-mer biotinylated oligonucleotide(Oligo 1, Tm=40° C.), in 1 mL of hybridization buffer, for one hour withgentle mixing. The coated beads were washed 5× with hybridization bufferat 60° C. and then resuspended in hybridization buffer at a finalconcentration of 10 ug/mL. The magnetic beads were then coated withlabeled mouse immunoglobulin by the following procedure: Mouseimmunoglobulin (mIgG) was labeled with Sulfo-TAG™ ECL labels (Meso ScaleDiagnostics, LLC.) according to the manufacturer's instructions. Theprotein was also labeled with an oligonucleotide having a terminal thiolgroup (Oligo 2, the complement of Oligo 1) using a bifunctional couplingreagent (sulfosuccinimidyl 4-(N-maléimidométhyl)-1-cyclohexanecarboxylate (“SMCC”)) and conventional coupling protocols, e.g., proteinis reacted with the NHS-ester in SMCC to label the protein and theresulting complex is reacted with thiolated oligonucleotides whichreacts with the maleimide group in SMCC. The labeled mIgG-oligoconjugate (0.1 pmol) was then mixed with the oligo-coated magnetic beads(500 ug of beads) in hybridization buffer for 1 hour at room temperatureto hybridize the complementary oligonucleotide sequences and therebyimmobilize the mIgG onto the beads. The resulting antibody-coated beadswere washed and resuspended in hybridization buffer.

The beads were incubated under different conditions, includingincubating the suspension at room temperature for one hour (with orwithout the presence of free Oligo2 as a competitor) and incubating thesuspension at 60′C for 10 min. (with or without the presence of freeOligo2 as a competitor). The beads were then magnetically collected andthe supernatant analyzed by ECL assay to measure the amount of labeledmIgG that was released from the beads. To measure the labeled mIgG, thesupernatant was transferred to the well of a MULTI-ARRAY plate in whichthe electrode is coated with goat anti-mouse antibodies (MULTI-ARRAY GAMPlate, Meso Scale Diagnostics, LLC.). The plate was incubated withshaking during which time labeled mIgG in the solution bound to theimmobilized goat anti-mouse antibodies. The wells were washed with PBS,filled with 150 uL of Read Buffer T (Meso Scale Diagnostics) andanalyzed on a SECTOR Imager instrument.

Table 1 shows that, in the absence of competing oligonucleotides, thelinkage of the mIgG to the beads was stable at room temperature. ThemIgG could be efficiently released from the beads by exposure to shortperiods of time above the melting temperature of the Oligo1-Oligo2 pair.The efficiency of release could be further enhanced by addition of freeOligo2 as a competitor.

TABLE 1 Efficiency of different release techniques. Release Technique %of Released Material 1 H at RT  6% 1 H at RT with free Oligo 23% 10 min60 C. 50% 10 min 60 C. with Free Oligo 57%

Example 4 Assay Including Capture of Analyte Through Collection ofMagnetic Particles and Release by Denaturation of a Linkage Comprisingan Oligonucleotide Pair

Magnetic beads (Dynalbeads® MyOne™-Streptavidin C1 beads, InvitrogenCorporation) were coated with biotinylated oligonucleotides as describedin Example 3. The magnetic beads were then coated with antibodiesagainst human TNF-alpha and IL-5 using i) antibodies that were labeledwith Sulfo-TAG and Oligo1 and ii) the coating procedure of Example 3.

Assay Procedure with Pre-Concentration. Sample containing humanTNF-alpha or IL-5 (1 mL of sample) was combined with 200 ng ofantibody-coated beads (prepared as described above) and incubated for 1hr at room temperature. The beads were magnetically collected and washedwith hybridization buffer. The antibody on the beads (including anylabeled-antibody-analyte complexes that were formed during theincubation) were released into 100 uL of a 1:20 dilution ofhybridization buffer (˜10 mM salt) at elevated temperature (60° C.),i.e., by denaturing the oligonucleotide pairs linking the antibodies tothe beads. The resulting solution was transferred to a well of aMULTI-ARRAY 96-well plate, each well of which included an array ofcapture antibodies including an anti-TNF-alpha spot and an anti-IL-5spot. The plate was incubated with shaking for 1 hr at room temperatureto allow the labeled-antibody-analyte complexes to bind to theappropriate capture antibody spots. The wells were then washed threetimes with PBS and then filled with 125 uL of Read Buffer T (Meso ScaleDiagnostics) and read on a SECTOR Imager instrument. The instrumentmeasures and reports the ECL intensity from each array element (or“spot”) in the antibody array.

Conventional Immunoassay Protocol without Pre-Concentration. Samplecontaining human TNF-alpha or IL-5 (30 uL) was combined with 20 uL, of asolution containing labeled (Sulfo-TAG) detection antibodies at aconcentration of 1 ug/mL. The resulting solution was incubated for 1 hrin a well of a MULTI-ARRAY plate having anti-TNF-alpha and anti-IL-5spots. The wells were washed, filled with Read Buffer T and analyzed ina SECTOR Imager instrument as described for the protocol with collectionand release.

Results. The results presented in Table 2 show that the protocol withcollection and release provided specific assay signals for bothTNF-alpha and IL-5 (signal in the presence of analyte—signal in theabsence of analyte) that were substantially higher than those obtainedusing the conventional protocol, without any substantial change in thebackground signal in the absence of analyte. The enhancement in specificsignal for 10 pg/mL samples was greater than 5-fold for TNF-alpha andgreater than 10-fold for IL-5.

TABLE 2 Assay Analyte TNF IL-5 Concentration, Conven- Pre- Conven- Pre-pg/mL tional Concentration tional Concentration 0 371 398 22 27 1 5301,095 95 451 10 3,301 18,323 723 9,831 100 31,005 75,864 8,057 48,895

Example 5 Multi-Well Plate

Another embodiment is a multi-well plate comprising at least one wellhaving (1) a binding surface having a first binding reagent immobilizedthereon and (2) at least one additional dry reagent, wherein at leastone additional dry reagent is a reconstitutable dry reagent that doesnot contact the binding surface. The multi-well plate may have anelectrode surface with a binding surface incorporated in at least onewell of the multi-well plate.

FIGS. 9a-9e show non-scale schematic views of several embodiments ofwell 100 of a multi-well plate. The well is defined by well floor 120and well walls 110. Floor 120 and walls 110 may be formed of a singlecontiguous material or may be separate components (e.g., a plate top andplate bottom) that are mated together. Well 100 also contains a firstdry reagent 130 located on floor 120 that, as shown, may be one or morecapture reagents that are immobilized on floor 120 to form a bindingsurface. First dry reagent 130 may include a plurality of immobilizedcapture reagents (e.g., reagents 130 a, 130 b, and 130 c) that arepatterned into a plurality of discrete binding domains (e.g., an array).Advantageously, the binding reagents/domains may have different affinityor specificity for binding partners; such binding domains may be used tocarry out multiplexed array-based measurements. A reconstitutableprotective layer 140 covers dry reagent 130. Protective layer 140 may beomitted, e.g., when it is not required to physically separate reagents130 and 150. Well 100 also comprises a second dry reagent 150 that is areconstitutable dry reagent. Second dry reagent 150 may comprise adetection reagent such as labeled detection reagent 160. Optionally,second dry reagent 150 comprises a plurality of detection reagents thatdiffer in affinity or specificity for binding partners. Well 100 mayalso include an, optional, additional reconstitutable dry reagent 170that comprises an assay control analyte 180 (as shown in FIGS. 9c-9e ).Also shown is plate seal 190. Plate seal 190, which may be omitted, issealed against the top surface of walls 110 to protect the dry reagentsfrom the environment.

FIG. 9a shows an embodiment in which first dry reagent 130 is coatedwith reconstitutable protective layer 140. Second dry reagent 150 islayered onto of protective layer 140 which prevents second dry reagent150 from contacting first dry reagent layer 130. In one example of thisembodiment, second dry reagent 150 is deposited by dispensing it inliquid form on protective layer 140; protective layer 140 is chosen tohave enough thickness or mass such that it can adsorb this liquidwithout allowing it to contact dry reagent 130. The liquid is then driedto form second dry reagent 150. In an alternate example, protectivelayer 140 is introduced in liquid form and frozen in the well to form afirst frozen layer. Reagent 150 is then introduced in liquid form andfrozen as a second frozen layer over the first frozen layer.Lyophilization of the two frozen layers provides the layered dry reagentstructure.

FIG. 9b shows an embodiment where reagents 130 and 150 are both fixedlylocated on non-overlapping regions of floor 120. Additional dryreagents, such as assay control reagents (not shown), could be locatedon other non-overlapping regions of floor 120. The localization ofreagents on selected regions of floor 120 may be carried out usingstandard techniques in patterned reagent deposition or dispensing.Optionally, floor 120 has relatively hydrophilic domains surrounded byrelatively hydrophobic areas such that appropriate volumes of reagentsdispensed on the hydrophilic domains will spread to defined boundariesdetermined by the hydrophobic areas. In this and other embodiments wherereconstitutable dry reagents are located on a surface, one may pre-treatthe surface with blocking agents to prevent adsorption of the reagentsto the surfaces and/or include blocking agents in the reagentcomposition.

FIG. 9c shows an embodiment where second dry reagent 150 is fixedlylocated, as one or more dry reagent pills, on walls 110. The pills maybe formed, e.g., by dispensing one or more droplets of the reagent (inliquid form) on walls 110 and drying them to form the dry reagent pills.FIG. 9c also shows optional additional dry reagent 170 with controlanalyte 180 fixedly located on another non-overlapping region of walls110, FIG. 9d shows an embodiment that is like that shown in FIG. 9cexcept that reagents 150 and 170 are located on shelves 115 on walls110. Dry reagents 150 and 170 may be formed from liquid reagents bydispensing and drying them on shelves 115 or dispensing them aboveshelves 115 so that they run down walls 110 onto shelves 115 where theyare dried. Alternatively, free-standing dry reagent pills may be placedon shelves 115.

Finally, FIG. 9e shows an embodiment where reagent 150 and optionalreagent 170 are free standing dry reagent pills. Also included areembodiments of well 100 in which there is some combination ofreconstitutable dry reagents on the well floor, well walls, wellshelves, and/or in free-standing form. In alternate embodiments, somecombination of fixedly located and free standing reconstitutable dryreagents is employed.

As shown in the embodiments in FIG. 9a-9e , the multi-well platesinclude those having wells with multiple, physically-distinct, dryreagents. Similarly, for carrying out different assays in differentwells, there may be different dry reagents in different wells. It may bedesirable, for example for QC purposes, to be sure that the correct dryreagents are present in the wells of a plate. Accordingly, the dryreagents may include indicators (such as dyes or fluorophores) that canbe used in optical inspection of the plates. By using differentdistinguishable indicators in different dry reagents, it is possible tooptically inspect a plate to ensure that the correct reagents are in theappropriate locations in the appropriate wells of a plate.

FIGS. 10a-10j shows non-scale schematic views of several embodiments ofwells that have shelf elements on which liquid reagents can be held anddried and/or on which free-standing dry reagents may be supported abovethe well bottom. The shelf elements may include ledges, bridges ortables as described below. FIG. 10a is a cross-section of a well 200showing well bottom 200 and well wall 210, the well wall having ledgessuch as ledges 230 and 235 that can support dry reagents. Ledge 230 hasan angle that is substantially 90° or less than 90° relative to the walldirectly above the ledge such that an appropriate volume of reagent canbe dispensed on ledge 230 and accumulate on ledge 230 withoutoverflowing onto well bottom 200. The ledges may also have additionalfeatures to help contain reagents such as lip 240 on ledge 235.

Shelf elements such as ledge 235 may be located at any height (h_(s))above well bottom 240 (h_(b)=0) and below the height of the well(h_(w)). In some embodiments, h_(s) is greater than or equal to 0.02h_(w), 0.05 h_(w) or 0.1 h_(w) but less than or equal to 0.1 h_(w), 0.25h_(w) or 0.5 h_(w). In other embodiments, h_(s) is greater or equal toabout 0.1 mm, 0.2 mm, 0.5 mm, or 1 mm but less than or equal to about 1mm, 2 mm, or 5 mm. Through proper selection of shelf height and volumesof sample/reagent added during the course of an assay, it may bepossible to control the order or timing of assay reactions. In oneexample, the shelf height and sample volume are chosen such thataddition of sample to the well provides a height of liquid that contactsreagents on the bottom of the well and also reconstitutes reagents onone or more shelves. Alternatively, shelf height may be chosen so thataddition of defined volume of a first liquid contacts dry reagents onthe bottom of the well (reconstituting reconstitutable reagents on thebottom and/or allowing reactions to proceed involving reagents stored onthe bottom) but does not reach the height of one or more shelves.Reactions involving reagents on the shelves can be commenced at a latertime by adding sufficient volume of a second liquid so that the liquidlevel reaches the height of the shelves so as to reconstitute dryreagent on the shelves, in conducting an assay, the sample to bemeasured may be the first liquid, second liquid or both.

FIGS. 10b-10f show top views of several embodiments of well 200 and showthat the well openings may have a variety of shapes including, but notlimited to, square (FIGS. 10b-10d ) and round (FIGS. 10e-10f ).Furthermore, the shelf elements may extend around the perimeter of thewell as in FIGS. 10b and 10e or there may be one or more isolated shelfelements that only extend partially around the well as in FIGS. 10c-20dand 10f . A well may also include a plurality of shelf elements atdifferent heights within a well. FIGS. 10 g-10 h show cross-section andtop views, respectively, of a well 290 in which a shelf element isprovided by bridge 250 that extends across the well. FIGS. 10i-10j showcross-section and top views, respectively of a well 295 in which a shelfelement I provided by a table 260 that extends vertically from an areaof well bottom 220.

A multi-well plate is provided comprising a plate body with a pluralityof wells defined therein including: a) a plurality of first reagentwells holding a reconstitutable first dry reagent and b) a plurality ofsecond reagent wells holding a second dry reagent (which may be areconstitutable dry reagent or an immobilized reagent), wherein, thefirst and second reagents are matched reagents for conducting an assay(i.e., they are both used in conducting an assay of interest). Thereagents may be located in a variety of locations with the wells such aswell bottom, well walls, on shelf elements, as free-standing pills orpowders, etc. A method is provided for carrying out assays in theseplates comprising: a) adding a sample to one of the first reagent wells,b) reconstituting reconstitutable dry labeled detection reagents in thefirst reagent well to form a reaction mixture, c) transferring analiquot of the reaction mixture to one or more of the second reagentwells, and d) incubating the reaction mixture in the second reagentwell(s) so as to carry out said assay on said sample. In one embodiment,the multi-well assay plate can be divided into a plurality of sets ofwells consisting of one first reagent well and one or more secondreagent wells and the method further comprises repeating the process of(a)-(d) for each set of wells.

FIG. 11a is a (not to scale) schematic illustration of one embodimentshowing cross-sectional views of two wells of a multi-well plate 300.Well 302 is a reagent reconstitution well comprising one or morereconstitutable dry reagents which may include a labeled detectionreagent (such as dry reagent 350 comprising labeled detection reagent360) or a an assay control analyte (such as dry reagent 370 comprisingassay control analyte 380). These dry reagents may include additionalreagent components such as blocking agents, stabilizers, preservatives,salts, pH buffers, detergents, bridging reagents, ECL coreactants andthe like. The reagents may be located on well bottoms, specificlocations on well bottoms, on well walls, shelf elements or may befree-standing (as per the discussion of FIGS. 9a-9e and 10a-10j ). Well301 is a detection well comprising one or more dry reagents which mayinclude reconstitutable dry reagents or an immobilized dry reagent. Asshown, well 301 comprises immobilized capture reagents 330 that arepatterned into three binding domains 330 a, 330 b, and 330 c to form abinding surface. Well 301 also comprises a reconstitutable protectivelayer 340 which may be omitted. In one embodiment of an assay, sample isadded to the reagent reconstitution well where reconstitutable dryreagents are reconstituted. The sample is then transferred to thedetection well where the assay measurement is carried out.Alternatively, a reconstitution buffer may be used to reconstitutereagents in the reagent reconstitution well; the reconstitution bufferis then combined with sample in the detection well. FIG. 11a also showsplate seal 390 which seals against the openings of wells 301 and 302 toprotect the contents of the wells from the environment.

The detection and reagent reconstitution wells in a multi-well plate maybe grouped into a plurality of assay sets consisting of one reagentreconstitution well and one or more detection wells, the reagentreconstitution well and detection wells within a set comprising matchedcapture and detection reagents for measuring an analyte of interest.FIG. 11b shows an arrangement where a set has one reagent reconstitutionwells 302 and three detection wells 301. During an assay, a sample addedto well 302 may then be distributed among the three associated detectionwells 301 so as to conduct multiple replicates or, where the detectionwells hold different reagents, multiple different assays. FIG. 11c showsan arrangement where a set has one reagent reconstitution well 302 andone detection well 301.

Reagent reconstitution wells and detection wells may be similar in sizeand shape or may have different sizes and/or shapes. In some embodiment,the wells in a standard multi-well plate are divided between the twotypes of wells. FIGS. 12a and 12b show a non-scale schematic views of analternative arrangement of wells. FIG. 12a shows a top view ofmulti-well plate 400 having detection wells 440 that are arranged in aregular two dimensional pattern and that have detection wells walls 430with inner wall surfaces and outer wall surfaces. Multi-well plate alsohas reagent reconstitution wells 460 in interstitial spaces betweendetection wells. Reagent reconstitution wells 460 have well walls thatare defined by the outer well surfaces of detection well walls 430 andrib elements 450 that connect the outer surfaces of well walls 430 ofadjacent detection wells (and, in the outermost of the wells, by theinner surface of plate frame wall 410). As shown, the detection wellsmay be shaped to have no reentrant (i.e., inward pointing) curves orangles while the interstitial wells may have reentrant curves and/orangles. FIG. 12b shows a cross-sectional view along the dotted line inFIG. 12a and shows the bottom surfaces of the two types of wells (whichmay be at different heights in the plate body). Plate 400 may be formedfrom a single contiguous material. In an alternate embodiment, plate 400is formed from a plate top 405 and a plate bottom 420 that are matedalong the dotted line shown in FIG. 12b . Advantageously, the basicarrangement of arrays of round wells with interstitial wells defined bythe well walls and rib elements is a common feature of manyinjection-molded 96-well plates and plate tops and allows thesecomponents to be used to form dry reagent plates as shown in FIGS. 12aand 12 b.

A multi-well plate is provided comprising a) a plate body with aplurality of wells defined therein including: i) a plurality of assaywells comprising a dry assay reagent; and ii) a plurality of desiccantwells comprising a desiccant, and b) a plate seal sealed against saidplate body thereby isolating said plurality of wells from the externalenvironment, some embodiments, the assay wells comprise the necessaryreagents for conducting an assay in the assay well. Also included areembodiments in which the desiccant wells are connected by dryingconduits to the assay wells, the conduits permitting diffusion of watervapor from the assay wells to the desiccant wells but intersecting thewells at a height in the assay well above the location of the dry assayreagent. In addition to multi-well plates containing dry reagents anddesiccants, the plates themselves (i.e., without dry reagents anddesiccants), in particular, plates having conduit or channel elements(e.g., as shown in FIGS. 13a-13f described below) that are suitable forconnecting sets of desiccant and assay wells with dry reagents areprovided.

FIGS. 13a-13f show non-scale schematic views of a multi-well plate 500having assay wells 501 and desiccant wells 502 (desiccant and dryreagents are not shown). FIG. 13a is a top view showing well walls 510and conduits 508 connecting dessicant wells with one (e.g., as in row A)or more assay wells (e.g., as in row B). FIG. 13b shows across-sectional view along the dotted line in FIG. 13a and together withFIG. 13a shows how conduits 508 may be formed by sealing plate seal 515against channels in the top surface of the plate body. Plate seal 515seals against these channels and the tops of the wells to form sets ofassay and dessicant wells that are interconnected by conduits but areisolated from the environment and from other sets of wells. Accordingly,one or more sets of wells may be unsealed and used in an assay and theremaining sets of wells will be maintained in a dry environmentallyprotected environment. Plate 500 may be formed from a single contiguousmaterial. In an alternate embodiment, plate 500 is formed from a platetop 505 and a plate bottom 512 that are mated along the dotted lineshown in FIG. 13b , plate bottom 512 defining the floor of at least someof the wells.

The assay wells or sets of wells in plate 500 may include one or more ofany of the dry reagent-containing wells described above, for example, inthe descriptions of FIGS. 9a-12b and may include both detection wellsand reagent reconstitution wells. The desiccants used in the desiccantwell may be selected from known desiccant materials including, but notlimited to, silica, activated alumina, activated clays, molecular sievesand other zeolites, hydroscopic salts (e.g., anhydrous calcium sulfate,magnesium sulfate, sodium sulfate, sodium hydroxide and lithiumchloride), hydroscopic solutions (e.g., concentrated solutions oflithium chloride) and water reactive materials such as phosphorouspentoxide. In some embodiments, the desiccant is present as a free drypowder or granular material. In other embodiments, the desiccant ispresent as a dry pill, for example a pressed tablet or a desiccantimpregnated polymeric material. In other embodiments, the desiccant iscontained in a water vapor permeable bag or container (e.g., as incommercial silica pouches). Advantageously, desiccant in pill form orpre-packaged containers may be “press fit” into desiccant wells toprevent movement in the well during shipping or use.

FIGS. 13c-13d show top and cross-sectional views of one embodiment of amulti-well plate 520 with assay and desiccant wells. Plate 520 has assaywells 521 (which may contain dry assay reagents) that are arranged in aregular two dimensional pattern and that have assay well walls 523 withinner wall surfaces and outer wall surfaces. It also has desiccant wells522 in interstitial spaces between detection wells (alternatively, wells521 are used as desiccant wells and wells 522 are used as assay wells).Desiccant wells 522 have well walls that are defined by the outer wellsurfaces of detection well walls 523 and rib elements 525 that connectthe outer surfaces of well walls 523 of adjacent assay wells (and, inthe outermost of the wells, by the inner surface of plate frame wall526). Channels 524 notched into the top of well walls 523 provide, whenmated to a plate seal, paths for water vapor to travel from assay wellsto desiccant wells. As shown, the assay wells may be shaped to have noreentrant (i.e., inward pointing) curves or angles while theinterstitial wells may have reentrant curves and/or angles. FIG. 13dshows a cross-sectional view along the dotted line in FIG. 13c and showsplate seal 527 which is mated to the top of the plate to form sets ofassay and desiccant wells that are connected via conduits 524 butisolated from other wells and from the environment. Plate 520 may beformed from a single contiguous material. In an alternate embodiment,plate 520 is formed from a plate top 528 and a plate bottom 529 that aremated along the dotted line shown in FIG. 13 d.

FIG. 13e shows a schematic view of another embodiment of a multi-wellplate with assay wells (which may contain dry reagents) and desiccantwells and shows a plate 540 with assay wells 541 and desiccant wells 543that are connected into sets of wells via channels 542 in the platebody. Multi-well plate 540 is largely analogous to the embodiment ofplate 500 pictured in FIGS. 13a-13b except that in plate 540, desiccantwells 542 are much shallower and smaller in area than the assay wellsallowing a larger portion of the plate footprint to be dedicated towells used in assay measurements. FIG. 13f shows a cross-sectional viewalong the dotted line in FIG. 13e and also shows plate seal 544 that issealed against the top of the plate to form connected sets of assay anddesiccant wells. Plate 540 may be formed from a single contiguousmaterial. In an alternate embodiment, plate 540 is formed from a platetop 545 and a plate bottom 546 that are mated along the dotted lineshown in FIG. 13f plate bottom 546 also defining the floor of assaywells 541.

FIG. 14 is a schematic exploded view of one embodiment of a multi-wellassay plate. Multi-well assay plate 600 comprises a plate top 610 withthrough-holes 615 that define the walls of wells. Plate top 610 issealed against plate bottom 620 through gasket 625 such that platebottom 620 defines the bottom surface of the wells. Optionally, platetop 610 is sealed directly to plate bottom 620 and gasket 625 isomitted. Sealing may be accomplished through traditional sealingtechniques such as adhesives, solvent welding, heat sealing, sonicwelding and the like. In another optional embodiment, plate top 610fully defines the sides and bottom of the wells and plate bottom 620 andgasket 625 may be omitted. The contents of the wells, which may includewells configured to contain dry reagent and/or desiccant as describedabove, may be protected from the outside environment by sealing (e.g.,via traditional sealing techniques) plate seal 630 to plate top 610directly or via optional gasket 635.

The components of plate 600 may be made from a variety of differentmaterials including, but not limited to, plastics, metals, ceramics,rubbers, glasses or combinations thereof. In accordance with therequirements of the particular detection technology used with theplates, the components some or all of the components may be selected tobe transparent, colored, opaque, or highly light scattering. In oneembodiment, plate top 610 is an injection-molded plastic such asinjection-molded polystyrene, polypropylene, or cyclic olefin copolymer(COC). Optionally, one or more of the components may be made of orcomprise (for example in the form of a coating) a material that has alow water vapor transmission rate, e.g., a water vapor transmission rateless than 1 g/m.sup.2 per day through a 100 um thickness. Low watervapor transmission materials include, but are not limited to, glass,metals or metal films (e.g., aluminum films), COC, polyvinylidenechloride (PvDC), polypropylene, polychlorotrifluoroethylene (PCTFE), andliquid crystal polymers (LCP).

Plate 600 may include desiccant wells as described above. Alternatively,or in addition, desiccant may be incorporated directly into plate top610, plate bottom 620, plate seal 630, gasket 625 and or gasket 635. Forexample, U.S. Pat. No. 6,174,952 to Hekal et al. describes desiccantcontaining polymer blends that may be molded, cast into liners, orformed into films, sheets, beads or pellets.

In some embodiments, plate bottom 620 has features to facilitate thepatterning of reagents on the bottom of wells (e.g., patternedhydrophilic features surrounded by hydrophobic areas) and/or conductivelayers that provide electrodes that are exposed to the interior volumesof the wells of plate 600 so that electrochemical or electrode inducedluminescence assays (e.g., electrochemiluminescence assays) may becarried out. Plate bottom 620 may also include electrode contacts toallow an external instrument to apply electrical potential/current tothe electrodes. Suitable approaches, configurations and compositions forsuch features, conductive layers and electrode contacts include thosedescribed in U.S. Publications 2004/0022677 and 2005/0052646 toWohlstadter et al. Suitable instrumentation and methods that can be usedto conduct ECL measurements using assay modules include those describedin U.S. Publications 2004/0022677 and 2005/0052646 of U.S. applicationSer. Nos. 10/185,274 and 10/185,363, respectively; U.S. Publication2003/0113713 of U.S. application Ser. No. 10/238,391; U.S. Publication2005/0142033 of U.S. application Ser. No. 10/980,198; and theconcurrently filed U.S. application Ser. No. 11/642,968 of Clinton etal. entitled “Assay Apparatuses, Methods and Reagents.”

FIGS. 15a-15c provide schematic illustrations of one specific embodimentthat includes some of the inventive concepts disclosed above in thecontext of a multi-well plate configured to carry out array-basedmultiplexed electrochemiluminescence assays. FIG. 15a shows a section ofmulti-well plate 700 that has a plurality of assay wells 710 which maycomprise dry reagents and a plurality of desiccant wells 720 which maycomprise a desiccant. Channels 725 on the top surface of plate 700 linkeach desiccant well to an assay well. Optionally, desiccant wells 720and channels 725 are omitted. Assay wells 710 have ledges 712 which maybe used to support a reconstitutable dry reagent (e.g., dry reagentscomprising assay controls and/or ECL labeled detection reagents). Assaywells also have working electrode surfaces 714 which are covered bypatterned dielectric layer 716 so as to expose a plurality of exposedelectrode surfaces or “spots” (shown as circles within the wells). Inaddition, counter electrodes 718 are provided to provide for a completeelectrochemical circuit. Optionally, the surface of dielectric layer 716is hydrophobic relative to electrode surface 714 so that small volumesof reagents patterned onto the spots may be kept confined to the spots.The different spots may have different capture reagents immobilizedthereon to form a binding surface with an array of binding domainsdiffering in specificity or affinity for binding partners (e.g.,analytes of interest). Alternatively, some of the spots may havereconstitutable dry reagents confined thereon which, e.g., may containassay controls and/or ECL labeled detection reagents. The assay well mayfurther comprise a reconstitutable protective layer over the bindingsurface.

FIG. 15b provides an exploded cross-sectional view along the dotted linein FIG. 15a and illustrates one approach to forming theelectrode/dielectric layers in assay wells 710. The multi-well platecomprises a plate top 730 that defines desiccant wells 720 and hasthrough-holes that define the walls of assay wells 710 and ledges 712.Plate top 730 also has channels 725 that form conduits between assaywells 710 and desiccant wells 720 when plate seal 750 is sealed againstthe top surface of plate top 730. In one non-limiting example, plate top730 is an injection-molded part molded from a plastic with low watervapor transmission. In another non-limiting example, plate seal 750 is aheat sealable film comprising a low water vapor transmission plastic ora metal (e.g., aluminum) foil.

FIG. 15b also shows plate bottom 740 which seals against plate top 730and defines the bottom of assay wells 710. Plate bottom 740 comprisessubstrate 715 which supports patterned conductive layers that providefor electrodes 714 and 718. Patterned dielectric layer 716 on theelectrodes defines the exposed electrode spots. A variety of materialsmay be used to provide for the substrate and the conductive anddielectric layers (see, e.g., U.S. Publications 2004/0022677 and2005/0052646). In one non-limiting example, the substrate is a plasticfilm (made, e.g., of a polyester such as MYLAR, polyvinylchloride, or alow water vapor transmissive material such as COC), the conductivelayers are screen printed conducting inks (e.g., screen printed carboninks) and the dielectric layer is a screen printed insulating ink. Alsoshown in FIG. 15b are electrode contacts 780 and 785 which areconductive layers on the bottom of substrate 715 that provideconnectivity (e.g., via conductive through holes in substrate 715 toelectrodes 714 and 718. The electrode contacts may also be provided byscreen printed conductive inks which during printing can be caused tofill holes in substrate 715 to also provide the conductivethrough-holes. Advantageously, the conductive through-holes may belocated directly below well walls to limit water vapor transmissionthrough the holes. In addition, an optional bottom sealing layer 790 maybe sealed to the bottom of substrate 715. Bottom sealing layer 790 ismade of a low water vapor transmissive material and covers most of thebottom surface of substrate 715 except for defined openings in sealinglayer 790 that are located so as to allow a plate reading instrument tocontact electrode contacts 780 and 785.

FIG. 15c shows a more detailed angled view of one embodiment of plate700 and shows desiccant pills 722 that are press-fit into desiccantwells 720.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of themethod in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

The invention claimed is:
 1. An assay device comprising: (a) a storage zone comprising a storage surface to which two or more surface-reagent complexes are bound and thereby confined to said storage zone, said two or more surface-reagent complexes comprising a first surface-reagent complex and a second surface-reagent complex, wherein said first surface-reagent complex comprises: (i) a first reagent linked to a first targeting agent, (ii) a first surface linked to a second targeting agent, and said second surface-reagent complex comprises: (iii) a second reagent linked to a third targeting agent, and (iv) a second surface linked to a fourth targeting agent, wherein said first surface and said second surface are portions of the same storage surface, wherein said first reagent and said first surface and said second reagent and said second surface are linked, respectively, in said first and second surface-reagent complexes, via a selective releasable binding interaction between said first and second targeting agents and said third and fourth targeting agents, respectively, wherein said first reagent is configured to release from said first surface-reagent complex by a first set of conditions, and said second reagent is configured to release from said second surface-reagent complex by a second set of conditions, wherein the first set of conditions is different from said second set of conditions; and (b) one or more use zones each configured to use at least one of said first reagent and said second reagent in an assay for an analyte of interest in a sample.
 2. The assay device of claim 1 wherein said one or more use zones each comprise an additional reagent used in said assay.
 3. The assay device of claim 1 wherein at least one of said first reagent and said second reagent are binding reagents that bind a component of said assay conducted in said use zone.
 4. The assay device of claim 3 wherein said at least one of said first reagent and said second reagent are binding reagents, and wherein said binding reagents bind said analyte.
 5. The assay device of claim 2 wherein said additional reagent binds said analyte.
 6. The assay device of claim 4, wherein said one or more use zones each comprise an additional reagent used in said assay, and wherein said at least one of said first reagent and said second reagent are binding reagents, and wherein said binding reagents bind a complex formed between said additional reagent and said analyte.
 7. The assay device of claim 2 wherein said one or more use zones each comprise a solid support and said additional reagent is bound to said solid support.
 8. The assay device of claim 1 wherein said at least one of said first and second reagents comprises a detectable label.
 9. The assay device of claim 8 wherein said detectable label is an ECL label.
 10. The assay device of claim 1 wherein said storage zone and said one or more use zones are in fluidic communication along a fluid path.
 11. The assay device of claim 1 wherein said one or more use zones each comprise two or more assay regions each configured to use one of said first and second reagents in one or more assays conducted with said sample in said assay device.
 12. The assay device of claim 11 wherein a first assay region of said one or more use zones is configured to conduct an assay for a first analyte of interest in said sample and an additional assay region in said one or more use zones is configured to conduct an assay for an additional analyte of interest in said sample.
 13. The assay device of claim 11 wherein a first assay region of said one or more use zones is configured to conduct a first assay for said analyte of interest in said sample and an additional assay region of said one or more use zones is configured to conduct a second assay for said analyte of interest in said sample.
 14. The assay device of claim 11 wherein each of said two or more assay regions comprise an additional reagent used in said assay.
 15. The assay device of claim 14 wherein said additional reagent is an additional binding reagent.
 16. The assay device of claim 11 wherein said one or more use zones each comprise an array of said two or more assay regions.
 17. The assay device of claim 1 wherein at least one of said first and second surfaces is a particle.
 18. The assay device of claim 1 wherein at least one of said first and second surfaces is roughened such that the surface area accessible to a component capable of binding to said surface is at least three-fold larger than the surface area of a flat surface.
 19. The assay device of claim 1 wherein at least one of said first and second surfaces is roughened such that the surface area accessible to a component capable of binding to said surface is at least two-fold larger than the surface area of a flat surface.
 20. The assay device of claim 1 wherein at least one of said first and second surfaces comprises a composite material including exposed particles distributed in a matrix.
 21. The assay device of claim 20 wherein said composite material comprises carbon particles, graphitic particles, or carbon nanotubes.
 22. The assay device of claim 20 wherein said composite is etched.
 23. The assay device of claim 20 wherein said surface comprises one or more indentations and/or raised features.
 24. The assay device of claim 1 wherein at least one of said first and second surfaces comprises a hydrogel.
 25. The assay device of claim 1 wherein said first set of conditions and said second set of conditions are selected from subjecting said storage zone to increased or decreased temperature, pH changes, an electric potential, a change in ionic strength, competition, and combinations thereof.
 26. The assay device of claim 25 wherein at least one of said first set of conditions and said second set of conditions is subjecting said storage zone to increased temperature.
 27. The assay device of claim 26 wherein said increased temperature exceeds the melting temperature of said binding interaction.
 28. The assay device of claim 1 wherein said assay device is a cartridge.
 29. The assay device of claim 1 wherein said assay device is a multi-well assay plate and said use zone is positioned within a well of said assay plate.
 30. The assay device of claim 29 wherein said storage zone is located on a supplemental surface of said well that does not overlap with said use zone.
 31. The assay device of claim 1 wherein said device is configured to conduct a multiplexed measurement. 